Methods and compositions for targeting polyubiquitin

ABSTRACT

Anti-K63-linked polyubiquitin monoclonal antibodies, and methods for using the antibodies, are provided.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional application No. 61/011,577, filed Jan. 18, 2008, and to U.S.provisional application No. 61/127,862, filed May 16, 2008, the contentsof which are incorporated in their entirety herein by reference.

FIELD OF THE INVENTION

This invention relates to the field of anti-polyubiquitin antibodies,and more particularly to anti-polyubiquitin antibodies that do notspecifically bind to monoubiquitin and that can discriminate betweenpolyubiquitins having different isopeptide linkages.

BACKGROUND

Ubiquitin is a small protein that has important regulatory roles in awide variety of cellular pathways. The best known of these isubiquitin's role in protein degradation, where covalent attachment ofubiquitin to a target protein enables that target protein to berecognized and destroyed by the 26S proteasome (see Wilkinson, Semin.Cell Devel. Biol. 11(3): 141-148 (2000)). Protein kinase regulation ofvarious signaling pathways has also been correlated with ubiquitination(see Sun and Chen, Curr. Opin. Cell Biol. 16: 119-126 (2004)). Forexample, phosphorylation of IκB by IκB kinase permits ubiquitination ofIκB and subsequent degradation by the 26S proteasome; because IκB is aninhibitor of NFκB, the degradation of IκB activates NFκB (Ghosh andKarin, Cell 109 (Suppl.): S81-S96 (2002); Palombella et al., Cell 78:773-785 (1994)). Ubiquitination also mediates DNA repair (see Sun andChen, Curr. Opin. Cell Biol. 16:119-126 (2004)). After DNA is damaged,monoubiquitination of proliferating cell nuclear antigen (PCNA)activates damage-tolerant polymerases which are able to synthesize DNAdespite any DNA lesions (Stelter and Ulrich, Nature 425: 188-191 (2003).Other physiological processes in which ubiquitination is known to beinvolved include cell division, cell growth, cell movement, andapoptosis/cell death (Johnson, Nat. Cell Biol. 4:E295-E298 (2002);Pickart, Mol. Cell. 8: 499-504 (2001)).

The covalent attachment of ubiquitin, a 76 amino acid protein, to atarget protein is a three-step enzymatic process (Pickart, Annu. Rev.Biochem. 70: 503-533 (2001)). First, ubiquitin-activating enzyme E1forms an ubiquitin-E1 thioester in an ATP-dependent reaction. Theubiquitin is transferred from the ubiquitin-E1 thioester to a member ofthe ubiquitin-conjugating enzyme (E2) family in the second step. In thethird step, with the assistance of a ubiquitin-protein ligase (E3), anisopeptide bond is formed between the carboxyl terminus of ubiquitin andthe ε-amino group of a lysine residue on the target protein. Enzymestermed deubiquitinases remove ubiquitin moieties from target proteins(Guterman and Glickman, Curr. Prot. Pep. Sci. 5: 201-210 (2004)).Highlighting ubiquitin's role as an important regulatory molecule, thehuman genome contains many different proteins involved in ubiquitinationor deubiquitination: at least 40 different E2s, 500 different E3s, and80 different deubiquitinases have been identified thus far (Wong et al.,Drug. Discov. Today 8: 746-754 (2003)).

Ubiquitin contains seven lysine residues (Lys6, Lys11, Lys27, Lys33,Lys29, Lys48, and Lys63), and thus ubiquitin itself may serve as atarget protein for ubiquitination (Peng et al., Nat. Biotechnol. 21:921-926 (2003); Pickart and Fushman, Curr. Opin. Chem. Biol. 8:610-616(2004)). The molecule produced upon ubiquitination of a ubiquitinprotein is termed a polyubiquitin molecule, and may comprise two or moreubiquitin moieties. Ubiquitination of ubiquitin may theoretically occurat any of the seven lysine residues (Peng et al., Nat. Biotechnol. 21:921-926 (2003)), so that different species of polyubiquitins existhaving isopeptide bonds to different lysine residues within ubiquitin.It is possible that a single polyubiquitin molecule with greater thantwo ubiquitin moieties may have more than one type of lysine linkage.Studies have shown that the E2 enzyme influences the type of lysinelinkage created between one ubiquitin molecule and another (Tenno etal., Genes to Cells 9: 865-875 (2004); Deng et al. (2000); Hofmann andPickart (2001)). Polyubiquitin and ubiquitin exist both as freemolecules and in covalent attachment with a target protein.

Like ubiquitin, polyubiquitin involvement has been found in manycellular processes, including intracellular trafficking, endocytosis,gene expression/silencing, proteolysis, kinase activation, translation,and DNA repair (Hoege et al., Nature 419:135-141 (2002); Spence et al.,Mol. Cell. Biol. 15:1265-1273 (1995); Hofmann and Pickart, Cell 96:645-653 (1999). Polyubiquitin and polyubiquitination can have strikinglydifferent physiological roles than monoubiquitin and monoubiquitinationin the same pathways, however. For example, whereas monoubiquitinationof PCNA after DNA damage results in the activation of error-prone DNApolymerases, polyubiquitination of PCNA at the identical residue wheremonoubiquitination is observed results in activation of error-free DNArepair (Stelter and Ulrich, Nature 425: 188-191 (2003); Hoege et al.,Nature 419:135-141 (2002); Spence et al., Mol. Cell. Biol. 15:1265-1273(1995); and Hofmann and Pickart, Cell 96: 645-653 (1999)).

Even polyubiquitins having different lysine linkages appear to playdifferent physiological roles. The two best-studied are the Lys48-linkedand Lys63-linked polyubiquitins, and structural studies of the twosuggest that different lysine-linked polyubiquitins may adopt markedlydifferent conformations, thus permitting different interactions withselected binding partners (Tenno et al., Genes to Cells 9: 865-875(2004)). Covalent modification by Lys48-linked polyubiquitin typicallymarks the target protein for proteolytic degradation, though there issome evidence that Lys48-linked polyubiquitin may also regulate certainproteins by non-proteolytic means (Chau et al., Science 243: 1576-1583(1989); Finley et al., Mol. Cell. Biol. 14: 5501-5509 (1994); Flick etal., Nat. Cell. Biol. 6:634-641 (2004)). Lys63-linked polyubiquitins, incontrast, have been linked to a variety of nonproteolytic intracellularpathways, including DNA repair (yeast cells expressing K63R-ubiquitinare defective in DNA repair), kinase activation, intracellulartrafficking, and translation (Pickart and Fushman, Curr. Opin. Chem.Biol. 8: 610-616 (2004); Hicke and Dunn, Annu Rev. Cell Dev. Biol. 19:141-172 (2003); Spece et al., Mol. Cell. Biol. 15: 1265-1273 (1995);Ulrich, Eukaryot. Cell 1: 1-10 (2002); Spence et al., Cell 102: 67-76(2000); Seibenhener et al., Mol. Cell. Biol. 24(18): 8055-8068 (2004)).In one specific example, synphilin-1 is normally ubiquitinated withK63-linked polyubiquitin by parkin in a proteasomal-independent manner,but synphilin-1 can alternately be targeted for destruction byubiquitination with K48-linked polyubiquitin (Lim et al., J. Neurosci.25(8): 2002-9 (2005)). An analysis of subjects with Parkinson's diseaseshows that K63-polyubiquitination of synphilin-1 may be involved in theformation of Lewy body inclusions associated with that disease (Lim etal., J. Neurosci. 25(8): 2002-9 (2005)).

Other lysine-linked polyubiquitins have not been studied extensively,largely because of the difficulty in distinguishing between them.Studies have thus far relied on cells expressing mutagenized ubiquitinsin which one or more lysines have been removed, on enzymaticallysynthesized polyubiquitins of particular linkages, or on techniques suchas mass spectrometry to distinguish between one type of polyubiquitinand another. Each of those methodologies is ill-suited or cumbersome foranalysis of the normal physiological behavior of particularlysine-linked polyubiquitins. While antibodies exist that are specificfor polyubiquitin as opposed to monoubiquitin (Fujimoro et al., FEBSLett. 349: 173-180 (1994)), there are as yet no antibodies that candistinguish between polyubiquitins of different lysine linkages.

Unsurprisingly, given their important roles in a variety of cellularprocesses, ubiquitin and polyubiquitins have also been implicated inmany diseases (see Argiles, Ubiquitin and Disease, R. G. Landes (1998)).Ubiquitin dysregulation is observed in muscle wasting (Mitch andGoldberg, New Engl. J. Med. 335: 1897-905 (1996); Bodine et al., Science294: 1704-1708 (2001)). Several genetic diseases have been linked toaberrant ubiquitin activity, including cystic fibrosis (Ward et al.,Cell 83: 121-127 (1995)), Angelman's syndrome (Kishino et al., NatureGenet. 15: 70-73 (1997)), and Liddle syndrome (Staub et al., EMBO J. 16:6325-6336 (1997)). Ubiquitin also plays a role in immune andinflammatory responses; for example, extracellular ubiquitin has beenfound to act as a sort of cytokine, inhibiting the TNFα response toendotoxin in peripheral blood mononuclear cells and regulating endotoxinhyporesponsiveness (Majetschak et al., Blood 101: 1882-1890 (2003);Ciechanover, EMBO J. 17: 7151-7160 (1998)). Also, both ubiquitin andpolyubiquitin have been found in human serum, with higher levels of bothmolecules observed in the serum of patients having parasitic andallergic disease (Takada et al., Clinical Chem. 43: 1188-1195 (1997)).

Dysregulation of several ubiquitin-mediated pathways are also involvedin cancer (Spataro et al., Br. J. Cancer 77: 448-55 (1998); Beckmann etal., Hum. Mutat. 25: 507-12 (2005)). For example, mutations in theheterodimeric ubiquitin ligase BRCA1-BARD1 are correlated with breastcancer (Hashizume et al., J. Biol. Chem. 276: 14537-40 (2001)),mutations that disrupt the ability of Myc to be degraded by theubiquitin pathway activate the oncogenic potential of c-Myc (Salghettiet al., EMBO J. 18: 717-726 (1999)), and transformed v-Jun is unable tobe ubiquitinated and degraded as its non-oncogenic correlate, c-Jun, is,giving rise to uncontrolled growth (Ciechanover, EMBO J. 17: 7151-7160(1998); Trier et al., Cell 78: 787-798 (1994)).

Ubiquitin and polyubiquitin have particularly been studied in thecontext of neurological diseases (Chung et al., TINS 24(11 Suppl.)S7-S14 (2001)). The inclusions, bodies, and neurofibrillary tangles thataccumulate in Huntington's disease, Spinocerebellar ataxia, prionencephalopathies, Pick's disease, Lewy body disease, Parkinson'sdisease, and Alzheimer's disease stain immunopositively for mono and/orpolyubiquitin (Alves-Rodrigues et al., Trends Neurosci. 21: 516-520(1998); Cammarata et al., Neurosci Lett. 156: 96-98 (1993); Kalchman etal., J. Biol. Chem. 271: 19385-94 (1996); Holmberg et al., Human Mol.Genet. 7: 913-918 (1998); Yedidia et al., EMBO J. 20: 5383-91 (2001);Mori et al., Science 235: 1641-44 (1987); Leigh et al., ActaNeuropathol.(Berl.) 79: 61-72 (1989); and Kuzuhara et al., ActaNeuropathologica 75:345-353 (1988)). Several forms of Parkinson's disease have been linkedto mutations in the ubiquitin carboxy-terminal hydrolase L1 (UCH-L1)gene, a deubiquitinase (Leroy et al., Nature 395: 451-452 (1998)), whileother forms of Parkinson's have been linked to inactivating mutations inParkin, an E2-dependent ubiquitin-protein ligase known to interact withthe ubiquitin-conjugating enzyme UbCH₇ and to ubiquitinate synphilin-1(Shimura et al., Nature Genet. 25: 302-305 (2000), Zhang et al., Proc.Natl. Acad. Sci. 97: 13354-13359 (2000); Lim et al., J. Neurosci. 25(8):2002-9 (2005)). Both types of mutations result in aberrant proteolyticprocessing and the inappropriate aggregation of proteins (see McNaughtet al., Nature Rev. Neurosci. 2: 589-594 (2001)). UCH-L1 mutations havealso been found to segregate with Huntington's disease (Naze et al.,Neurosci. Lett. 328: 1: 1-4 (2002)). A mutant form of ubiquitin has beenidentified in the brains of Alzheimer's patients that is veryefficiently incorporated into polyubiquitin chains, but is refractory todeubiquitination once formed, potentially leading to dominant inhibitionof the normal cellular proteolytic processing system (Lam et al., Proc.Natl. Acad. Sci. 97: 9902-9906 (2000)).

It is clear that it would be beneficial not only to have compositionsand methods that can distinguish between polyubiquitins of differentlysine linkages, but also to have compositions and methods that areeffective in targeting and modulating ubiquitin andpolyubiquitin-mediated pathways. The invention provided herein relatesto such compositions and methods.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention provides novel antibodies capable of binding to and/orregulating biological activities associated with polyubiquitin.

In one embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof that specifically binds to K63-linkedpolyubiquitin, wherein the antibody does not specifically bind toK48-linked polyubiquitin or monoubiquitin. In one aspect, the inventionprovides an isolated antibody or antigen-binding fragment comprising atleast one hypervariable (HVR) sequence selected from HVR-H2 and HVR-L2of any of SEQ ID NOs: 59-110 and 112, and 7-58 and 111, respectively.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, whereinthe antibody or antigen-binding fragment comprises at least one HVR-H2sequence, wherein HVR-H2 comprises the amino acid sequence a b c d e f gh i j k l m n o p (SEQ ID NO: 221), and wherein amino acid a is selectedfrom the amino acids tyrosine, aspartic acid and tryptophan; amino acidb is isoleucine; amino acid c is selected from the amino acids serine,threonine, alanine, phenylalanine, tyrosine, and valine; amino acid d isproline; amino acid e is tyrosine; amino acid f is selected from theamino acids tyrosine, phenylalanine, leucine, and histidine; amino acidg is glycine; amino acid h is selected from the amino acids serine,glycine, alanine, phenylalanine, and tryptophan; amino acid I isthreonine; amino acid j is serine; amino acid k is tyrosine; amino acidl is alanine; amino acid m is aspartic acid; amino acid n is serine;amino acid o is valine, and amino acid p is lysine.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody does not specifically bind toK48-linked polyubiquitin or monoubiquitin, and wherein the antibody orantigen-binding fragment comprises at least one HVR-H2 sequence selectedfrom the HVR-H2 sequences of SEQ ID NOs: 59-110 and 112. In one aspect,the antibody or antigen-binding fragment comprises at least one HVR-H2sequence selected from the HVR-H2 sequences of SEQ ID NOs: 60, 63, and66.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, andwherein the antibody or antigen-binding fragment comprises at least oneHVR-L2 sequence, wherein HVR-L2 comprises the amino acid sequence q r st u v w x (SEQ ID NO: 222), wherein amino acid q is selected from theamino acids tyrosine and phenylalanine; amino acid r is selected fromthe amino acids alanine and serine; amino acid s is alanine; amino acidt is selected from the amino acids serine, arginine, valine, threonine,alanine, asparagine, and leucine; amino acid u is serine; amino acid vis leucine; amino acid w is tyrosine, and amino acid x is serine.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, andwherein the antibody or antigen-binding fragment comprises at least oneHVR-L2 sequence selected from the HVR-L2 sequences of SEQ ID NOs: 7-58and 111. In one aspect, the antibody or antigen-binding fragmentcomprises at least one HVR-L2 sequence selected from the HVR-L2sequences of SEQ ID NOs: 8, 11, and 14.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, whereinthe antibody or antigen-binding fragment comprises at least one sequenceselected from HVR-H2 and HVR-L2, wherein HVR-H2 comprises the amino acidsequence a b c d e f g h i j k l m n o p (SEQ ID NO: 221), wherein aminoacid a is selected from the amino acids tyrosine, aspartic acid andtryptophan; amino acid b is isoleucine; amino acid c is selected fromthe amino acids serine, threonine, alanine, phenylalanine, tyrosine, andvaline; amino acid d is proline; amino acid e is tyrosine; amino acid fis selected from the amino acids tyrosine, phenylalanine, leucine, andhistidine; amino acid g is glycine; amino acid h is selected from theamino acids serine, glycine, alanine, phenylalanine, and tryptophan;amino acid I is threonine; amino acid j is serine; amino acid k istyrosine; amino acid l is alanine; amino acid m is aspartic acid; aminoacid n is serine; amino acid o is valine, and amino acid p is lysine;and wherein HVR-L2 comprises the amino acid sequence q r s t u v w x(SEQ ID NO: 222), wherein amino acid q is selected from the amino acidstyrosine and phenylalanine; amino acid r is selected from the aminoacids alanine and serine; amino acid s is alanine; amino acid t isselected from the amino acids serine, arginine, valine, threonine,alanine, asparagine, and leucine; amino acid u is serine; amino acid vis leucine; amino acid w is tyrosine, and amino acid x is serine.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, whereinthe antibody or antigen-binding fragment comprises at least one HVR-H2sequence and at least one HVR-L2 sequence, wherein HVR-H2 comprises theamino acid sequence a b c d e f g h i j k l m n o p (SEQ ID NO: 221),wherein amino acid a is selected from the amino acids tyrosine, asparticacid and tryptophan; amino acid b is isoleucine; amino acid c isselected from the amino acids serine, threonine, alanine, phenylalanine,tyrosine, and valine; amino acid d is proline; amino acid e is tyrosine;amino acid f is selected from the amino acids tyrosine, phenylalanine,leucine, and histidine; amino acid g is glycine; amino acid h isselected from the amino acids serine, glycine, alanine, phenylalanine,and tryptophan; amino acid I is threonine; amino acid; is serine; aminoacid k is tyrosine; amino acid l is alanine; amino acid m is asparticacid; amino acid n is serine; amino acid o is valine, and amino acid pis lysine; and wherein HVR-L2 comprises the amino acid sequence q r s tu v w x (SEQ ID NO: 222), wherein amino acid q is selected from theamino acids tyrosine and phenylalanine; amino acid r is selected fromthe amino acids alanine and serine; amino acid s is alanine; amino acidt is selected from the amino acids serine, arginine, valine, threonine,alanine, asparagine, and leucine; amino acid u is serine; amino acid vis leucine; amino acid w is tyrosine, and amino acid x is serine.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, whereinthe antibody or antigen-binding fragment comprises at least one HVR-H2sequence selected from the HVR-H2 sequences of SEQ ID NOs: 59-110 and112 and at least one HVR-L2 sequence selected from the HVR-L2 sequencesof SEQ ID NOs: 7-58 and 111.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, whereinthe antibody or antigen-binding fragment comprises at least one HVR-H2sequence selected from the HVR-H2 sequences of SEQ ID NOs: 60, 63, and66 and at least one HVR-L2 sequence selected from the HVR-L2 sequencesof SEQ ID NOs: 8, 11, and 14.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that specifically binds to K63-linkedpolyubiquitin, wherein the antibody or antigen-binding fragment does notspecifically bind to K48-linked polyubiquitin or monoubiquitin, whereinthe antibody or antigen-binding fragment comprises HVR amino acidsequences selected from the HVR-H2 and HVR-L2 amino acid sequences setforth for any one of Apu3.A8-Apu3.H10 in Table B. In one aspect, the HVRamino acid sequences are selected from the HVR-H2 and HVR-L2 amino acidsequences set forth for any one of Apu3.A8, Apu3.A12, and Apu3.B3 inTable B.

In one aspect, any of the foregoing antibody or antigen-bindingfragments comprise at least one HVR sequence selected from the HVR-H1sequence of SEQ ID NO: 5, the HVR-H3 sequence of SEQ ID NO: 6, theHVR-L1 sequence of SEQ ID NO: 3 and the HVR-L3 sequence of SEQ ID NO: 4.In another aspect, any of the foregoing antibody or antigen-bindingfragments comprise at least two HVR sequences selected from the HVR-H1sequence of SEQ ID NO: 5, the HVR-H3 sequence of SEQ ID NO: 6, theHVR-L1 sequence of SEQ ID NO: 3 and the HVR-L3 sequence of SEQ ID NO: 4.In another aspect, any of the foregoing antibody or antigen-bindingfragments comprise at least three HVR sequences selected from the HVR-H1sequence of SEQ ID NO: 5, the HVR-H3 sequence of SEQ ID NO: 6, theHVR-L1 sequence of SEQ ID NO: 3 and the HVR-L3 sequence of SEQ ID NO: 4.In another aspect, any of the foregoing antibody or antigen-bindingfragments comprise the HVR-H1 sequence of SEQ ID NO: 5, the HVR-H3sequence of SEQ ID NO: 6, the HVR-L1 sequence of SEQ ID NO: 3 and theHVR-L3 sequence of SEQ ID NO: 4.

In one aspect, any of the foregoing antibody or antigen-bindingfragments have an affinity for K63-linked polyubiquitin improvedrelative to the affinity of the parental Fab Apu2.16 for K63-linkedpolyubiquitin. In another aspect, any of the foregoing antibody orantigen-binding fragments have K_(d) values for K63-linked polyubiquitinless than or equal to 10 nM.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that binds to the same antigenic determinant onpolyubiquitin as any of the foregoing antibody or antigen-bindingfragments, wherein the antibody or antigen-binding fragment does notspecifically bind to monoubiquitin. In another embodiment, the inventionprovides an isolated antibody or antigen-binding fragment that competeswith any of the foregoing antibody or antigen-binding fragments forbinding to polyubiquitin, wherein the antibody or antigen-bindingfragment does not specifically bind to monoubiquitin.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment that binds to an epitope in K63-linkedpolyubiquitin. In one aspect, the epitope includes residues in both afirst ubiquitin subunit and a second ubiquitin subunit of the K63-linkedpolyubiquitin. In another aspect, the epitope includes at least oneresidue in a first ubiquitin subunit selected from Glu-18, Pro-19,Ser-20, Asp-21, Thr-55, Leu-56, Ser-57, Asp-58, Asn-60, Ile-61, andGln-62. In another aspect, the epitope includes at least one residue ina second ubiquitin subunit selected from Leu-8, Thr-9, Glu-34, Gly-35,Ile-36, Pro-37, Asp-39, Gln-40, Leu-71, Arg-72, Leu-73, Arg-74, andGly-75. In another aspect, the epitope includes at least one residue ina first ubiquitin subunit selected from Glu-18, Pro-19, Ser-20, Asp-21,Thr-55, Leu-56, Ser-57, Asp-58, Asn-60, Ile-61, and Gln-62, and at leastone residue in a second ubiquitin subunit selected from Leu-8, Thr-9,Glu-34, Gly-35, Ile-36, Pro-37, Asp-39, Gln-40, Leu-71, Arg-72, Leu-73,Arg-74, and Gly-75. In another aspect, the N-terminal portion of HVRH3contacts C-terminal residues and Q40 from the donor ubiquitin. Inanother aspect, the distal part of HVRH3 contacts the 50s loop of theK63-acceptor ubiquitin. In another such aspect, the N-terminal portionof HVRH3 contacts C-terminal residues 72-74 and Q40 from the donorubiquitin, while residues R102 and Y103 of HVRH3 contact the 50s loop ofthe K63-acceptor ubiquitin. In another aspect, C-terminal residues ofthe donor ubiquitin contact the antibody or antigen-binding fragment. Inanother such aspect, the donor ubiquitin residues involved in thecontact are L73 and R74.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment comprising improved electrostatic compatibilitybetween its light chain and the surface of a K63-acceptor ubiquitin. Inone aspect, the antibody or antigen-binding fragment comprises an Arg atposition 52 of HVRL2. In another aspect, the antibody or antigen-bindingfragment comprises an Arg at position 66 of the light chain frameworkregion. In another aspect, the antibody or antigen-binding fragmentcomprises an Arg at position 52 of HVRL2 and an Arg at position 66 ofthe light chain framework region.

In another embodiment, any of the foregoing antibodies orantigen-binding fragments specifically binds to a K63-linkedpolyubiquitinated protein. In one aspect, the antibody orantigen-binding fragment inhibits degradation of the K63-linkedpolyubiquitinated protein. In another aspect, the antibody orantigen-binding fragment modulates at least one polyubiquitin-mediatedsignaling pathway. In another aspect, the antibody or antigen-bindingfragment inhibits at least one polyubiquitin-mediated signaling pathway.In another aspect, the antibody or antigen-binding fragment stimulatesat least one polyubiquitin-mediated signaling pathway.

In another embodiment, the invention provides a nucleic acid moleculeencoding any of the foregoing antibody or antigen-binding fragments. Inanother embodiment, the invention provides a vector comprising such anucleic acid molecule. In another embodiment, the invention provides ahost cell comprising such a vector. In another embodiment, the inventionprovides a cell line capable of producing any of the foregoingantibodies or antigen-binding fragments. In another embodiment, theinvention provides a method of producing any of the foregoing antibodiesor antigen-binding fragments, comprising culturing a host cellcomprising a nucleic acid molecule encoding the antibody orantigen-binding fragment under conditions wherein the antibody orantigen-binding fragment is produced.

In another embodiment, the invention provides a composition comprisingan effective amount of any of the foregoing antibodies orantigen-binding fragments and a pharmaceutically acceptable carrier. Inone aspect, the composition comprises two or more of the foregoingantibodies or antigen-binding fragments.

In another embodiment, the invention provides a method of identifyingthe presence of K63-linked polyubiquitin or a K63-linkedpolyubiquitinated protein in a sample, comprising contacting the samplewith at least one of the foregoing antibodies or antigen-bindingfragments.

In another embodiment, the invention provides a method for the treatmentof a disease or condition associated with dysregulation of polyubiquitinin a patient, the method comprising administering to the patient aneffective amount of at least one of the foregoing antibodies orantigen-binding fragments. In one aspect, the patient is a mammalianpatient.

In another aspect, the patient is human. In another aspect, the diseaseis selected from cancer, a muscular disorder, aubiquitin-pathway-related genetic disorder, an immune/inflammatorydisorder, and a neurological disorder. In another aspect, the disease isselected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, musculardystrophy, multiple sclerosis, amyotrophic lateral sclerosis, cysticfibrosis, Angelman's syndrome, Liddle syndrome, Alzheimer's disease,Parkinson's disease, Pick's disease, and Paget's disease.

In another embodiment, the invention provides a method of determiningthe presence of a K63-linked polyubiquitin or a K63-linkedpolyubiquitinated protein in a sample suspected of containing aK63-linked polyubiquitin or K63-linked polyubiquitinated protein,comprising exposing the sample to at least one of the foregoingantibodies or antigen-binding fragments and determining the binding ofthe at least one antibody or antigen-binding fragment to a K63-linkedpolyubiquitin or K63-linked polyubiquitinated protein in the sample.

In another embodiment, the invention provides a method of separatingK63-linked polyubiquitinated protein from non-K63-linkedpolyubiquitinated protein in a sample, comprising contacting the samplewith at least one of the foregoing antibodies or antigen-bindingfragments.

In another embodiment, the invention provides a method of determiningthe function and/or activity of K63-linked polyubiquitin in a cellcomprising contacting the cell with at least one of the foregoingantibodies or antigen-binding fragments and assessing the effect of saidcontacting step on the cell. In another embodiment, the inventionprovides a method of determining the function and/or activity ofK63-linked polyubiquitin in a sample comprising contacting the samplewith at least one of the foregoing antibodies or antigen-bindingfragments and assessing the effect of said contacting step on thesample.

An antibody of the invention can be in any number of forms. For example,an antibody of the invention can be a chimeric antibody, a humanizedantibody or a human antibody. In one embodiment, an antibody of theinvention is not a human antibody, for example it is not an antibodyproduced in a xenomouse (e.g., as described in WO96/33735). An antibodyof the invention can be full length or a fragment thereof (e.g., afragment comprising an antigen binding component).

In another embodiment, the invention provides an antigen-bindingfragment of any of the above-described antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the primary structure of ubiquitin and schematic views ofcertain polyubiquitin isopeptide linkages. FIG. 1A shows the amino acidsequence of human ubiquitin (SEQ ID NO: 223), with the lysine residuesindicated in bold, underlined text. FIG. 1B shows a schematic depictionof the bond formed between the lysine-48 or lysine-63 of a firstubiquitin molecule and the C-terminal glycine residue of a secondubiquitin molecule.

FIGS. 2A-E depict crystal structures of relevant molecules as discussedin Example 4. FIG. 2A depicts the complex between K63-linked diubiquitin(upper part of figure) and the Apu2.16 Fab fragment (lower part offigure, with the light chain on the left and the heavy chain on theright), and shows that the heavy chain CDR3 (“H3”) contacts bothubiquitin chains on either side of the isopeptide linkage. H3 side chainwithin 4.2 Å of diubiquitin, and ubiquitin side chains within 4.2 Å ofH3, are shown as sticks. Residues labeled in bold are ubiquitin residueswhile the other (non-bold) labeled residues are Fab residues. K63 in theacceptor ubiquitin is shown as a sphere. FIG. 2B depicts a comparison ofK63-linked (upper) and K48-linked (lower) diubiquitin structures. Inboth cases, the lysine acceptor ubiquitin is to the left and the donorubiquitin is to the right of the figure. The K48-linked diubiquitinforms a more compact shape with the chain extending perpendicular to theubiquitin dimer as compared to the K63 dimer, where the chain extends ina more elongated manner. FIG. 2C is a superimposition of the Apu2.16crystal structure shown in FIG. 2A superimposed on the crystal structureof Apu3.A8, showing the location of the two changes in L2 (S52R) and H3(S52T) introduced during the affinity maturation process to createApu3.A8. FIG. 2D shows a comparison of the structures of Apu2.16,Apu3.A8 and a variant of humanized 4D5 (pdb 1FVE). The Fv regions ofApu2.16 and Apu3.A8 are shown as tubes and superimposed on the Fv regionof the humanized anti-Her2 antibody 4D5 variant. In the top view, theCDR regions are labeled; in the bottom view, the Fv regions are rotatedby 90 degrees to show the N-termini. FIG. 2E depicts the chargecomplementarity between Apu3.A8 (lower) and diubiquitin (upper).Electrostatic surfaces were calculated with PyMol. Regions of positivepotential are shaded; regions with negative potential are shaded andindicated with dotted lines.

FIGS. 3A-D depict the results of western blotting experiments describedin Example 1(E). FIG. 3A shows the binding of the parentalanti-K63-linked polyubiquitin Fab Apu2.16 to immobilized K63-linkeddiubiquitin and the absence of binding to immobilized K48-linkeddiubiquitin. FIG. 3B shows the binding of anti-K63-linked polyubiquitinFab Apu3.A8 to immobilized K63-linked diubiquitin and the absence ofbinding to immobilized K48-linked diubiquitin. FIG. 3C shows the bindingof anti-K63-linked polyubiquitin Fab Apu3.A 12 to immobilized K63-linkeddiubiquitin and the absence of binding to immobilized K48-linkeddiubiquitin. FIG. 3D shows the binding of anti-K63-linked polyubiquitinFab Apu3.B3 to immobilized K63-linked diubiquitin and the absence ofbinding to immobilized K48-linked diubiquitin.

FIG. 4 depicts the results of western blotting experiments described inExample 2. The figure shows the poor binding of the parental Apu2.16 IgGto either immobilized K63-linked di- to heptaubiquitin or immobilizedK48-linked di- to heptaubiquitin. The figure also shows the binding ofeach of the affinity-matured anti-K63-linked polyubiquitin antibodiesApu3.A12, Apu3.B3, and Apu3.A8 to immobilized K63-linked di- toheptaubiquitin and the absence of binding to immobilized K48-linked di-to heptaubiquitin.

FIG. 5 depicts the results of western blotting experiments described inExample 2. The figure shows that though the parental anti-K63-linkedpolyubiquitin antibody Apu2.16 was not able to detect K63-linkedubiquitinated Traf6, the affinity-matured antibodies Apu3.A12, Apu3.B3,and Apu3.A8 were able to specifically detect K63-linked ubiquitinatedTraf6 in a western blot format.

FIGS. 6A-B depict the results of a western blotting experiment to assessthe ability of various anti-K63-linked polyubiquitin antibodies toimmunoprecipitate K63-linked Traf6 from cellular lysates, as describedin Example 2. The affinity-matured antibodies Apu3.A8, Apu3.B3, andApu3.A12 were better able to immunoprecipitate K63-linkedpolyubiquitinated Traf6 than the parental antibody Apu2.16.

FIGS. 7A-K depict the results of confirmatory mass spectrometryexperiments, as described in Example 3. FIG. 7A depicts the results ofmass spectrometry experiments to confirm that the proteinsimmunoprecipitated by affinity-matured antibodies Apu3.A8, Apu3.B3, andApu3.A12 were mainly K63-linked ubiquitinated, as described in Example2. FIG. 7B shows that the K48 linkage was not enriched using thisapproach, hence showing specificity of the K63 antibody towards the K63chain linkage. FIGS. 7C-F show bar graphs of data obtained fromimmunoprecipitation experiments using anti-K48-linked polyubiquitinantibody Apu2.07, anti-K63-linked polyubiquitin antibody Apu3.A8, or anisotype control antibody (anti-Her2), followed by mass spectrometricanalysis to determine the total amount of ubiquitin immunoprecipitated(FIG. 7C), as well as the types of polyubiquitin linkages present in thelysate (FIG. 7D) and antibody-specific immunoprecipitates(anti-K48-linked polyubiquitin in FIG. 7E; anti-K63-linked polyubiquitinin FIG. 7F). FIG. 7G schematically depicts the MuRF1 autoubiquitinationreactions performed in vitro with WT, K48R or K63R ubiquitin followed byimmunoprecipitation with Apu2.07, Apu3.A8, or an isotype-matched controlantibody. The numbers in parentheses indicate the relevant lanes andcolumns in FIGS. 7H-K. FIG. 7H shows a western blot including thereactions depicted schematically in FIG. 7G. The blot was probed with apan-ubiquitin antibody. The horizontal dotted lines indicate the portionof the gel that was cut out and subjected to analysis by massspectrometry. FIGS. 7I-K show bar graphs of the mass spectrometry dataobtained from the experiments to determine the polyubiquitin linkages inthe autoubiquitination reactions and immunoprecipitations depictedschematically in FIG. 7G.

FIG. 8A schematically depicts the signaling pathway stimulated by tumornecrosis factor alpha (TNFα) binding to tumor necrosis factor receptor 1(TNFR1) in vivo. FIG. 8B schematically depicts the signaling pathwaystimulated by IL-1β binding to IL-1R1 in vivo.

FIG. 9A shows western blots from immunoprecipitation experiments todetect the ubiquitination state of RIP, as described in Example 3. Forthe purpose of describing this figure only, the blots are assignedsequential numbers from 1 to 7, with the topmost blot having assignednumber 1 and the bottom-most blot having assigned number 7. The blot 6includes samples that were immunoprecipitated with anti-K48-linked IgGto capture K48-linked polyubiquitinated proteins. The blot 7 includessamples that were immunoprecipitated with a 1:1 cocktail of Apu3.A8 andApu3.B3 to capture K63-linked polyubiquitinated proteins. Both blotswere stained with an anti-RIP antibody. The blots 1-3 show controlwestern blots to demonstrate that the levels of RIP and tubulin remainrelatively constant during TNFα treatment (blots 1 and 3), while IκBAlevels decrease upon TNFα treatment (blot 2). Blots 4 and 5 show controlwestern blots to demonstrate that RIP is co-precipitated during animmunoprecipitation for TNFR1 (blot 4), and that the levels of TNFR1remain constant during TNFα treatment (blot 5). FIG. 9B schematicallydepicts the cellular pathway by which K63-linked polyubiquitin is addedto RIP and replaced with K48-linked polyubiquitin by A20.

FIG. 10 shows the results of experiments described in Example 3(B)assessing the polyubiquitination state of IRAK1 after IL-1β stimulationof cells. Total IRAK1, IκBα and tubulin levels are shown in FIGS. 10Aand 10B. FIG. 10C shows western blots assessing IRAK1 that is modifiedby K48-linked polyubiquitin (top panel) or K63-linked polyubiquitin(lower panel). FIG. 10D shows a western blot indicating the effect ofthe protease inhibitor MG-132 on degradation of polyubiquitinated IRAK1.

FIG. 11 depicts immunofluorescence microscopy images of HeLa cellsstained with an anti-K48-linked polyubiquitin antibody or ananti-K63-linked polyubiquitin antibody alone (FIG. 11A or FIG. 11D,respectively) or further including a polyclonal antibody recognizing 20Sproteasome subunits (FIGS. 11B and 11E, respectively), as described inExample 5. The arrow indicates mid-body staining. In the merged images(FIGS. 11C and 11F), very bright staining indicates potentialcolocalization and less bright staining corresponds to DAPI-labelednuclei. The bars in each figure represent 50 μm.

FIGS. 12A and 12B and FIG. 13 depict exemplary acceptor human consensusframework sequences for use in practicing the instant invention withsequence identifiers as follows:

-   -   Variable heavy (VH) consensus frameworks (FIGS. 12A and 12B)

Human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NOs:113-116)

Human VH subgroup I consensus framework minus extended hypervariableregions (SEQ ID NOs: 117-128)

Human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NOs:129-132)

Human VH subgroup II consensus framework minus extended hypervariableregions (SEQ ID NOs: 133-144)

Human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID NOs:145-148)

Human VH subgroup III consensus framework minus extended hypervariableregions (SEQ ID NOs: 149-160)

Human VH acceptor framework minus Kabat CDRs (SEQ ID NOs: 161-164)

Human VH acceptor framework minus extended hypervariable regions (SEQ IDNOs: 165-172)

Human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NOs: 173-176)

Human VH acceptor 2 framework minus extended hypervariable regions (SEQID NOs: 177-188)

-   -   Variable light (VL) consensus frameworks (FIG. 13)

Human VL kappa subgroup I consensus framework (SEQ ID NOs: 189-192)

Human VL kappa subgroup II consensus framework (SEQ ID NOs: 193-196)

Human VL kappa subgroup III consensus framework (SEQ ID NOs: 197-200)

Human VL kappa subgroup IV consensus framework (SEQ ID NOs: 201-204)

FIG. 14 depicts framework region sequences of huMAb4D5-8 light and heavychains. Numbers in superscript/bold indicate amino acid positionsaccording to Kabat.

FIG. 15 depicts modified/variant framework region sequences ofhuMAb4D5-8 light and heavy chains. Numbers in superscript/bold indicateamino acid positions according to Kabat.

MODES FOR CARRYING OUT THE INVENTION General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, third edition (Sambrook et al., 2001);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); PCR 2: A Practical Approach (M.J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Harlow andLane, eds. (1988) Antibodies, A Laboratory Manual; “A Practical Guide toMolecular Cloning” (Perbal Bernard V., 1988); and “Phage Display: ALaboratory Manual” (Barbas et al., 2001).

DEFINITIONS

As used herein, the terms “ubiquitin” and “monoubiquitin” are usedinterchangeably, and are defined as all species of native human andsynthetic ubiquitin substantially similar to a 76-amino acid proteinhaving at least one lysine residue at amino acid 6, amino acid 11, aminoacid 27, amino acid 29, amino acid 33, amino acid 48, and/or amino acid63.

As used herein, the term “polyubiquitin” is defined as all species ofnative human and synthetic polymeric chains of ubiquitin which fallwithin human and synthetic classes of different polymeric linkages ofubiquitin, including, but not limited to, K6-linked polyubiquitin,K11-linked polyubiquitin, K27-linked polyubiquitin, K29-linkedpolyubiquitin, K33-linked polyubiquitin, K48-linked polyubiquitin andK63-linked polyubiquitin. Polyubiquitin may be of any length, andincludes at least two ubiquitin moieties. Polyubiquitin is distinguishedfrom tandem repeats of ubiquitin that are originally expressed as asingle protein.

As used herein, the terms “K*-linked polyubiquitin” and “Lys*-linkedpolyubiquitin” are interchangeable, and refer to a polyubiquitinmolecule comprising at least one isopeptide bond between the C-terminusof one ubiquitin moiety and a lysine at position * in another ubiquitinmoiety. For example, a “K63-linked polyubiquitin” is usedinterchangeably with a “Lys63-linked polyubiquitin”, and both termsrefer to a polyubiquitin molecule comprising an isopeptide bond betweenthe C-terminus of one of the ubiquitin moieties in the molecule and thelysine at position 63 in another ubiquitin moiety in the molecule.

As used herein, a statement that a first lysine linkage “differs” from asecond lysine linkage indicates that the first lysine linkage betweenone ubiquitin moiety and another ubiquitin moiety involves a differentlysine residue (e.g., K6, K11, K27, K29, K33, K48, and/or K63) than thesecond lysine linkage between one ubiquitin moiety and another ubiquitinmoiety.

As used herein, the term “ubiquitin pathway” refers to a biochemicalpathway in a cell or reconstituted in vitro that includes ubiquitinand/or polyubiquitin.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In one embodiment, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined by,for example, the Lowry method, and in some embodiments more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

As used herein, the terms “anti-ubiquitin antibody” and“anti-monoubiquitin antibody” are used interchangeably, and refer to anantibody that is capable of specifically binding to a ubiquitinmolecule.

As used herein, the term “anti-polyubiquitin antibody” refers to anantibody that is capable of specifically binding to a polyubiquitinmolecule.

As used herein, the term “anti-K48-linked polyubiquitin antibody” refersto an antibody that is capable of specifically binding to K48-linkedpolyubiquitin.

As used herein, the term “anti-K63-linked polyubiquitin antibody” refersto an antibody that is capable of binding to K63-linked polyubiquitin.

The phrase “substantially similar,” “substantially the same”,“equivalent”, or “substantially equivalent”, as used herein, denotes asufficiently high degree of similarity between two numeric values (forexample, one associated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said values (e.g., Kd values,anti-viral effects, etc.). The difference between said two values is,for example, less than about 50%, less than about 40%, less than about30%, less than about 20%, and/or less than about 10% as a function ofthe value for the reference/comparator molecule.

The phrase “substantially reduced,” or “substantially different”, asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of an anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., 65 hours) to insure that equilibriumis reached. Thereafter, the mixtures are transferred to the captureplate for incubation at room temperature (e.g., for one hour). Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5Ill/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. In each experiment, a spot wasactivated and ethanolamine blocked without immobilizing protein, to beused for reference subtraction. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g.,Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds10⁶ M¹s⁻¹ by the surface plasmon resonance assay above, then the on-ratecan be determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with astirred cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgram. The equilibrium dissociationconstant (Kd) was calculated as the ratio k_(off)/k_(on). See, e.g.,Chen, Y., et al., (1999)J. Mol Biol 293:865-881. However, if the on-rateexceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stirred cuvette.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle-stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, monovalent,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be chimeric, human, humanized and/oraffinity matured.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of heavy or light chain of the antibody. Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called a, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably, to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise an antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Thus, the modifier “monoclonal” indicates the characterof the antibody as not being a mixture of discrete antibodies. Suchmonoclonal antibody typically includes an antibody comprising apolypeptide sequence that binds a target, wherein the target-bindingpolypeptide sequence was obtained by a process that includes theselection of a single target binding polypeptide sequence from aplurality of polypeptide sequences. For example, the selection processcan be the selection of a unique clone from a plurality of clones, suchas a pool of hybridoma clones, phage clones or recombinant DNA clones.It should be understood that the selected target binding sequence can befurther altered, for example, to improve affinity for the target, tohumanize the target binding sequence, to improve its production in cellculture, to reduce its immunogenicity in vivo, to create a multispecificantibody, etc., and that an antibody comprising the altered targetbinding sequence is also a monoclonal antibody of this invention. Incontrast to polyclonal antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody of a monoclonal antibody preparation isdirected against a single determinant on an antigen. In addition totheir specificity, the monoclonal antibody preparations are advantageousin that they are typically uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by a variety oftechniques, including, for example, the hybridoma method (e.g., Kohleret al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2^(nd) ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage display technologies (See, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO98/24893; WO96/34096; WO96/33735;WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551(1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann etal., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016;

Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol.13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 or 49-56 (L2) and 89-97 or89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102,94-102, or 95-102 (H3) in the VH. The variable domain residues arenumbered according to Kabat et al., supra, for each of thesedefinitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or HVR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO93/1161; and Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies are produced by procedures known inthe art. Marks et al. Bio/Technology 10:779-783 (1992) describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofCDR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with anantibody of the invention. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include cancer, muscular disorders,ubiquitin-pathway-related genetic disorders, immune/inflammatorydisorders, neurological disorders, and other ubiquitin pathway-relateddisorders.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, leukemia and other lymphoproliferative disorders, andvarious types of head and neck cancer.

The term “muscular disorder” refers to or describes the physiologicalcondition in muscle-containing animals that is typically characterizedby deterioration or weakening of skeletal and/or smooth muscle such thatnormal muscular function is significantly reduced. Examples of musculardisorders include, but are not limited to, muscular dystrophy, multiplesclerosis, amyotrophic lateral sclerosis, Isaac's syndrome; stiff-personsyndrome; familiar periodic paralyses, myopathy, myotonia,rhabdomyolyses, muscle atrophy, and various types of muscle weakness andmuscle rigidity.

The term “ubiquitin pathway-related genetic disorder” refers to ordescribes a genetically-based disorder that is typically characterizedby or contributed to by aberrant functioning of the ubiquitin pathway.Examples of ubiquitin pathway-related genetic disorders include, but arenot limited to, cystic fibrosis, Angelman's syndrome, and Liddlesyndrome.

The terms “neurological disorder” or “neurological disease” refer to ordescribe a disease or disorder of the central and/or peripheral nervoussystem in mammals that is typically characterized by deterioration ofnervous tissue or deterioration of communication between cells innervous tissue. Examples of neurological disorders include, but are notlimited to, neurodegenerative diseases (including, but not limited to,Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome,olivopontocerebellar atrophy, Parkinson's disease, multiple systematrophy, striatonigral degeneration, tauopathies (including, but notlimited to, Alzheimer disease and supranuclear palsy), prion diseases(including, but not limited to, bovine spongiform encephalopathy,scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), bulbar palsy, motor neuron disease, andnervous system heterodegenerative disorders (including, but not limitedto, Canavan disease, Huntington's disease, neuronalceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkeskinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome,lafora disease, Rett syndrome, hepatolenticular degeneration,Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia(including, but not limited to, Pick's disease, and spinocerebellarataxia.

The terms “inflammatory disorder” and “immune disorder” refer to ordescribe disorders caused by aberrant immunologic mechanisms and/oraberrant cytokine signaling. Examples of inflammatory and immunedisorders include, but are not limited to, autoimmune diseases,immunologic deficiency syndromes, and hypersensitivity. An “autoimmunedisease” herein is a non-malignant disease or disorder arising from anddirected against an individual's own tissues. The autoimmune diseasesherein specifically exclude malignant or cancerous diseases orconditions, especially excluding B cell lymphoma, acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemiaand chronic myeloblastic leukemia. Examples of autoimmune diseases ordisorders include, but are not limited to, inflammatory responses suchas inflammatory skin diseases including psoriasis and dermatitis (e.g.atopic dermatitis); systemic scleroderma and sclerosis; responsesassociated with inflammatory bowel disease (such as Crohn's disease andulcerative colitis); respiratory distress syndrome (including adultrespiratory distress syndrome; ARDS); dermatitis; meningitis;encephalitis; uveitis; colitis; glomerulonephritis; allergic conditionssuch as eczema and asthma and other conditions involving infiltration ofT cells and chronic inflammatory responses; atherosclerosis; leukocyteadhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus(SLE) (including but not limited to lupus nephritis, cutaneous lupus);diabetes mellitus (e.g. Type I diabetes mellitus or insulin dependentdiabetes mellitus); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis;Sjogren's syndrome; juvenile onset diabetes; and immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes typically found in tuberculosis, sarcoidosis,polymyositis, granulomatosis and vasculitis; pernicious anemia(Addison's disease); diseases involving leukocyte diapedesis; centralnervous system (CNS) inflammatory disorder; multiple organ injurysyndrome; hemolytic anemia (including, but not limited to cryoglobinemiaor Coombs positive anemia); myasthenia gravis; antigen-antibody complexmediated diseases; anti-glomerular basement membrane disease;antiphospholipid syndrome; allergic neuritis; Graves' disease;Lambert-Eaton myasthenic syndrome; pemphigoid bullous; pemphigus;autoimmune polyendocrinopathies; Reiter's disease; stiff-man syndrome;Behcet disease; giant cell arteritis; immune complex nephritis; IgAnephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP)or autoimmune thrombocytopenia, etc.

Examples of immunologic deficiency syndromes include, but are notlimited to, ataxia telangiectasia, leukocyte-adhesion deficiencysyndrome, lymphopenia, dysgammaglobulinemia, HIV or deltaretrovirusinfections, common variable immunodeficiency, severe combinedimmunodeficiency, phagocyte bactericidal dysfunction,agammaglobulinemia, DiGeorge syndrome, and Wiskott-Aldrich syndrome.Examples of hypersensitivity include, but are not limited to, allergies,asthma, dermatitis, hives, anaphylaxis, Wissler's syndrome, andthrombocytopenic purpura.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing or decreasing inflammation and/or tissue/organdamage, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder.

An “individual” is a vertebrate. In certain embodiments, the vertebrateis a mammal. Mammals include, but are not limited to, farm animals (suchas cows), sport animals, pets (such as cats, dogs, and horses),primates, mice and rats. In certain embodiments, the vertebrate is ahuman.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. In certainembodiments, the mammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the substance/molecule are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolyticenzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosed below.Other cytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin aberrant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Compositions and Methods of Making Same

The present invention provides antibodies that bind specifically topolyubiquitin, but not to monoubiquitin. More specifically, antibodiesthat are capable of binding specifically to a polyubiquitin comprising aK63 linkage but not to a polyubiquitin comprising a second, different,lysine linkage are provided. The antibodies of the invention providesurprisingly improved affinity for K63-linked polyubiquitin overpreviously known antibodies. This improved affinity enables their broaduse in a variety of assays where tight binding of the antibody toK63-linked polyubiquitinated protein is a prerequisite, such asimmunoprecipitation reactions. The antibodies of the invention are alsobetter suited for use as therapeutics than previously identifiedanti-K63-linked polyubiquitin-specific antibodies because they maypotentially be administered at lower dosages or less frequently than anantibody with a lesser affinity for the same K63-linked polyubiquitin.

In one aspect, the invention provides an antibody comprising an HVR-H2region comprising the sequence of at least one of SEQ ID NOs: 60-110. Inone aspect, the invention provides an antibody comprising an HVR-H2region consensus sequence of SEQ ID NO: 112.

In one aspect, the invention provides an antibody comprising an HVR-L2region comprising the sequence of at least one of SEQ ID NOs: 8-58. Inone aspect, the invention provides an antibody comprising a HVR-L3region consensus sequence of SEQ ID NO: 111.

In one aspect, the invention provides an antibody comprising at leastone or at least two of the following:

(i) an HVR-H2 sequence comprising at least one sequence of SEQ ID NOs:60-110;

(ii) an HVR-L2 sequence comprising at least one sequence of SEQ ID NOs:8-58.

In one aspect, the invention provides an antibody that specificallybinds K63-linked polyubiquitin with high affinity but binds K48-linkedpolyubiquitin with substantially reduced affinity, comprising at leastone or at least two of the following:

(i) an HVR-H2 sequence comprising at least one sequence of SEQ ID NOs:60-110;

(ii) an HVR-L2 sequence comprising at least one sequence of SEQ ID NOs:8-58.

The amino acid sequences of SEQ ID NOs: 8-112 are numbered with respectto individual HVR (i.e., H2, L2) as indicated in Table B, the numberingbeing consistent with the Kabat numbering system as described below. Inone embodiment, an antibody of the invention comprises one or two of theHVR sequences of (i)-(ii) above, and at least one of HVR-L1 of SEQ IDNO: 3, HVR-L3 of SEQ ID NO: 4, HVR-H1 of SEQ ID NO: 5, and HVR-H3 of SEQID NO: 6.

In one aspect, the invention provides antibodies comprising heavy chainHVR H2 sequences as set forth in Table B. In one embodiment, theantibodies further comprise light chain HVR-L2 sequences as set forth inTable B.

Some embodiments of antibodies of the invention comprise a light chainvariable domain of humanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN®,Genentech, Inc., South San Francisco, Calif., USA) (also referred to inU.S. Pat. No. 6,407,213 and Lee et al., J. Mol. Biol. (2004),340(5):1073-93) as depicted in SEQ ID NO: 224 below.

(SEQ ID NO: 224) Asp¹ Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 

 Val 

 Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 

 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys¹⁰⁷ (HVR residues areunderlined)In one embodiment, the huMAb4D5-8 light chain variable domain sequenceis modified at one or more of positions 28, 30, 31, 42, 53, 66, and91-96 (Asp, Asn, Thr, Lys, Phe, Arg, His, Tyr, Thr, Thr, Pro, and Pro asindicated in bold/italics above, respectively). In one embodiment, themodified huMAb4D5-8 sequence comprises Ser in position 28, Ser inposition 30, Ser in position 31, Glu in position 42, Ser in position 53,Gly in position 66, Tyr in position 91, Ser in position 92, Ser inposition 93, Tyr in position 94, Ser in position 95, Ser in position 96,and/or an insertion of Leu and Phe between the Ser at position 96 andthe Thr at position 97. In another embodiment, the modified huMAb4D5-8sequence comprises an inserted Ser just prior to position 1(i.e., at theN-terminus of the protein), a Ser in position 28, Ser in position 30,Ser in position 31, Glu in position 42, Ser in position 53, Gly inposition 66, Tyr in position 91, Ser in position 92, Ser in position 93,Tyr in position 94, Ser in position 95, Ser in position 96, and/or aninsertion of Leu and Phe between the Ser at position 96 and the Thr atposition 97. Accordingly, in one embodiment, an antibody of theinvention comprises a light chain variable domain comprising thesequence depicted in SEQ ID NO: 225 below:

(SEQ ID NO: 225) Ser⁻¹ Asp¹ Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 

 Val 

 Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 

 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys (HVR residues areunderlined)Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics above.

Certain embodiments of antibodies of the invention comprise a heavychain variable domain having the following sequence:

(SEQ ID NO: 226) Glu Ile Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe AsnVal Lys Thr Gly Leu Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val Ala Tyr Ile 

 Pro Tyr Tyr Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr IleSer Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg AlaGlu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Tyr Tyr Arg Trp Tyr ThrAla Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (HVRresidues are underlined)In one embodiment, the heavy chain variable domain sequence is modifiedat the bold, italicized position. In another embodiment, the bold,italicized Ser is modified to Thr. In another embodiment, the firstthree residues of the heavy chain variable domain sequence set forth inSEQ ID NO: 226 are not present.

In another embodiment, an antibody of the invention comprises a heavychain variable domain having the following sequence:

(SEQ ID NO: 227) Glu Ile Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe AsnVal Lys Thr Gly Leu Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val Ala Tyr Ile 

 Pro Tyr Tyr Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr IleSer Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg AlaGlu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Tyr Tyr Arg Trp Tyr ThrAla Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (HVRresidues are underlined)

In another embodiment, an antibody of the invention comprises a heavychain variable domain having the following sequence:

(SEQ ID NO: 228) Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe AsnVal Lys Thr Gly Leu Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val Ala Tyr Ile 

 Pro Tyr Tyr Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr IleSer Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg AlaGlu Asp Thr Ala Val Tyr Tyr Cys Ala ArgGlu Tyr Tyr Arg Trp Tyr Thr Ala Ile Asp Tyr Trp Gly Gln Gly Thr Leu ValThr Val Ser Ser (HVR residues are underlined)

Antibodies of the invention can comprise any suitable framework variabledomain sequence, provided binding activity to polyubiquitin including aparticular lysine linkage is substantially retained. For example, insome embodiments, antibodies of the invention comprise a human subgroupIII heavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73 and/or 78. In some embodiments of these antibodies,position 71 is A, 73 is T and/or 78 is A. In one embodiment, theseantibodies comprise heavy chain variable domain framework sequences ofhuMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South San Francisco, Calif.,USA) (also referred to in U.S. Pat. Nos. 6,407,213 & 5,821,337, and Leeet al., J. Mol. Biol. (2004), 340(5):1073-93). In one embodiment, theseantibodies further comprise a human κI light chain framework consensussequence. In one embodiment, these antibodies comprise at least one, twoor more of the light chain HVR sequences of SEQ ID NOs: 3, 4, 8-58, and111. In one embodiment, these antibodies comprise light chain HVRsequences of huMAb4D5-8 as described in U.S. Pat. Nos. 6,407,213 &5,821,337. In one embodiment, these antibodies comprise light chainvariable domain sequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc.,South San Francisco, Calif., USA) (also referred to in U.S. Pat. Nos.6,407,213 & 5,821,337, and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93).

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequenceof at least one of SEQ ID NOs: 113-188, 209-212, and 217-220, the HVR H1sequence is SEQ ID NO: 5, the HVR H2 sequence is selected from at leastone of SEQ ID NOs: 59-110 and 112; and the HVR H3 sequence is SEQ ID NO:6. In one embodiment, an antibody of the invention comprises a lightchain variable domain, wherein the framework sequence comprises thesequence of at least one of SEQ ID NOs: 189-204, 205-208, and 213-216,the HVR-L1 sequence is SEQ ID NO: 3, the HVR-L2 sequence is selectedfrom at least one of SEQ ID NOs: 8-58 and 111, and the HVR-L3 sequenceis SEQ ID NO: 4.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises at least onesequence of SEQ ID NOs: 113-188, and HVR H1, H2 and H3 sequences are SEQID NOs: 5, 60, and 6, respectively (clone Apu3.A8). Similarly, in otherembodiments, antibodies of each of clones Apu3.A9-H10 as set forth onTable B herein comprise a heavy chain variable domain, wherein theframework sequence comprises at least one sequence of SEQ ID NOs:113-188, and HVR-H1 is SEQ ID NO: 5, HVR-H2 sequences are thosesequences specifically enumerated for each clone in Table B, and HVR-H3is SEQ ID NO: 6. In one embodiment, an antibody of the inventioncomprises a light chain variable domain, wherein the framework sequencecomprises at least one sequence of SEQ ID NOs: 189-204, and HVR L1, L2and L3 sequences are SEQ ID NOs: 3, 8, and 4, respectively (Apu3.A8).Similarly, in other embodiments, antibodies of each of clonesApu3.A9-H10 as set forth on Table B herein comprise a light chainvariable domain, wherein the framework sequence comprises at least onesequence of SEQ ID NOs: 189-204, and HVR-L1 is SEQ ID NO: 3, HVR-L2sequences are those sequences specifically enumerated for each clone inTable B, and HVR-L3 is SEQ ID NO: 4.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises at least onesequence of SEQ ID NOs: 209-212, and HVR H1, H2 and H3 sequences are SEQID NOs: 5, 60, and 6, respectively (clone Apu3.A8). Similarly, in otherembodiments, antibodies of each of clones Apu3.A9-H10 comprise a heavychain variable domain, wherein the framework sequence comprises at leastone sequence of SEQ ID NOs: 209-212, and HVR-H1 is SEQ ID NO: 5, HVR-H2sequences are those sequences specifically enumerated for each clone inTable B, and HVR-H3 is SEQ ID NO: 6. In one embodiment, an antibody ofthe invention comprises a light chain variable domain, wherein theframework sequence comprises at least one sequence of SEQ ID NOs:205-208, and HVR L1, L2 and L3 sequences are SEQ ID NOs: 3, 8, and 4,respectively (Apu3.A8). Similarly, in other embodiments, antibodies ofeach of clones Apu3.A9-H10 shown in Table B comprise a light chainvariable domain, wherein the framework sequence comprises at least onesequence of SEQ ID NOs: 205-208, and HVR-L1 is SEQ ID NO: 3, HVR-L2sequences are those sequences specifically enumerated for each clone inTable B, and HVR-L3 is SEQ ID NO: 4.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises at least onesequence of SEQ ID NOs: 217-220, and HVR H1, H2 and H3 sequences are SEQID NOs: 5, 60, and 6, respectively (clone Apu3.A8). Similarly, in otherembodiments, antibodies of each of clones Apu3.A9-Hi 0 comprise a heavychain variable domain, wherein the framework sequence comprises at leastone sequence of SEQ ID NOs: 217-220, and HVR-H1 is SEQ ID NO: 5, HVR-H2sequences are those sequences specifically enumerated for each clone inTable B, and HVR-H3 is SEQ ID NO: 6. In one embodiment, an antibody ofthe invention comprises a light chain variable domain, wherein theframework sequence comprises at least one sequence of SEQ ID NOs:213-216, and HVR L1, L2 and L3 sequences are SEQ ID NOs: 3, 8, and 4,respectively (Apu3.A8). Similarly, in other embodiments, antibodies ofeach of clones Apu3.A9-H10 shown in Table B comprise a light chainvariable domain, wherein the framework sequence comprises at least onesequence of SEQ ID NOs: 213-216, and HVR-L1 is SEQ ID NO: 3, HVR-L2sequences are those sequences specifically enumerated for each clone inTable B, and HVR-L3 is SEQ ID NO: 4.

In another example, an affinity matured antibody of the invention whichspecifically binds to K63-linked polyubiquitin with high affinity butbinds to K48-linked polyubiquitin with substantially reduced affinitycomprises substitution at HVR-H2 amino acid positions 50, 52, 53, 54 and56. In another example, an affinity matured antibody of the inventionwhich specifically binds to K63-linked polyubiquitin with high affinitybut binds to K48-linked polyubiquitin with substantially reducedaffinity comprises substitution at HVR-L2 amino acid positions 49, 50,52, and 53.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NOs: 5, 8, and 6. Inone embodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NOs: 3, 60, and 4. Inone embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NOs: 5, 8, and 6 andalso comprises a light chain variable domain comprising the sequence ofSEQ ID NOs: 3, 60, and 4. In another embodiment, an antibody of theinvention comprises a heavy chain variable domain comprising thesequence of SEQ ID NOs: 5, 63, and 6. In one embodiment, an antibody ofthe invention comprises a light chain variable domain comprising thesequence of SEQ ID NOs: 3, 11, and 4. In one embodiment, an antibody ofthe invention comprises a heavy chain variable domain comprising thesequence of SEQ ID NOs: 5, 63, and 6 and also comprises a light chainvariable domain comprising the sequence of SEQ ID NOs: 3, 11, and 4. Inanother embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NOs: 5, 66, and 6. Inone embodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NOs: 3, 14, and 4. Inone embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NOs: 5, 66, and 6 andalso comprises a light chain variable domain comprising the sequence ofSEQ ID NOs: 3, 14, and 4.

In one aspect, the invention provides an antibody that competes with anyof the above-mentioned antibodies for binding to polyubiquitin. In oneaspect, the invention provides an antibody that binds to the sameantigenic determinant on polyubiquitin as any of the above-mentionedantibodies.

As shown herein, the antibodies of the invention specifically bind to anisolated polyubiquitin having a specific lysine linkage. As shownherein, the antibodies of the invention also specifically bind topolyubiquitin having a K63 lysine linkage when that polyubiquitin isattached to a heterologous protein (see, e.g., Examples 2 and 3).

Compositions comprising at least one anti-polyubiquitin antibody or atleast one polynucleotide comprising sequences encoding ananti-polyubiquitin antibody are provided. In certain embodiments, acomposition may be a pharmaceutical composition. As used herein,compositions comprise one or more antibodies that bind to one or morepolyubiquitin and/or one or more polynucleotides comprising sequencesencoding one or more antibodies that bind to one or more polyubiquitin.These compositions may further comprise suitable carriers, such aspharmaceutically acceptable excipients including buffers, which are wellknown in the art.

Isolated antibodies and polynucleotides are also provided. In certainembodiments, the isolated antibodies and polynucleotides aresubstantially pure.

In one embodiment, anti-polyubiquitin antibodies are monoclonal. Inanother embodiment, fragments of the anti-polyubiquitin antibodies(e.g., Fab, Fab′-SH and F(ab′)2 fragments) are provided. These antibodyfragments can be created by traditional means, such as enzymaticdigestion, or may be generated by recombinant techniques. Such antibodyfragments may be chimeric, humanized, or human. These fragments areuseful for the diagnostic and therapeutic purposes set forth below.

Generation of Anti-Polyubiquitin Antibodies Using a Phage DisplayLibrary

A variety of methods are known in the art for generating phage displaylibraries from which an antibody of interest can be obtained. One methodof generating antibodies of interest is through the use of a phageantibody library as described in Lee et al., J. Mol. Biol. (2004),340(5): 1073-93.

The anti-polyubiquitin antibodies of the invention can be made by usingcombinatorial libraries to screen for synthetic antibody clones with thedesired activity or activities. In principle, synthetic antibody clonesare selected by screening phage libraries containing phage that displayvarious fragments of antibody variable region (Fv) fused to phage coatprotein. Such phage libraries are panned by affinity chromatographyagainst the desired antigen. Clones expressing Fv fragments capable ofbinding to the desired antigen are adsorbed to the antigen and thusseparated from the non-binding clones in the library. The binding clonesare then eluted from the antigen, and can be further enriched byadditional cycles of antigen adsorption/elution. Any of theanti-polyubiquitin antibodies of the invention can be obtained bydesigning a suitable antigen screening procedure to select for the phageclone of interest followed by construction of a full lengthanti-polyubiquitin antibody clone using the Fv sequences from the phageclone of interest and suitable constant region (Fc) sequences describedin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g. asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g. as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-polyubiquitin clones is desired, the subject isimmunized with polyubiquitin to generate an antibody response, andspleen cells and/or circulating B cells or other peripheral bloodlymphocytes (PBLs) are recovered for library construction. In oneembodiment, a human antibody gene fragment library biased in favor ofanti-human polyubiquitin clones is obtained by generating an anti-humanpolyubiquitin antibody response in transgenic mice carrying a functionalhuman immunoglobulin gene array (and lacking a functional endogenousantibody production system) such that polyubiquitin immunization givesrise to B cells producing human antibodies against polyubiquitin. Thegeneration of human antibody-producing transgenic mice is described inSection (II)(b) below.

Additional enrichment for anti-polyubiquitin reactive cell populationscan be obtained by using a suitable screening procedure to isolate Bcells expressing polyubiquitin-specific membrane bound antibody, e.g.,by cell separation with polyubiquitin affinity chromatography oradsorption of cells to fluorochrome-labeled polyubiquitin followed byflow-activated cell sorting (FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in whichpolyubiquitin is not antigenic. For libraries incorporating in vitroantibody gene construction, stem cells are harvested from the subject toprovide nucleic acids encoding unrearranged antibody gene segments. Theimmune cells of interest can be obtained from a variety of animalspecies, such as human, mouse, rat, lagomorpha, luprine, canine, feline,porcine, bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, the library diversity is maximized byusing PCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

Screening of the libraries can be accomplished by any art-knowntechnique. For example, polyubiquitin can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other art-known method forpanning phage display libraries.

The phage library samples are contacted with immobilized polyubiquitinunder conditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by polyubiquitinantigen competition, e.g. in a procedure similar to the antigencompetition method of Clackson et al., Nature, 352: 624-628 (1991).Phages can be enriched 20-1,000-fold in a single round of selection.Moreover, the enriched phages can be grown in bacterial culture andsubjected to further rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, forpolyubiquitin. However, random mutation of a selected antibody (e.g. asperformed in some of the affinity maturation techniques described above)is likely to give rise to many mutants, most binding to antigen, and afew with higher affinity. With limiting polyubiquitin, rare highaffinity phage could be competed out. To retain all the higher affinitymutants, phages can be incubated with excess biotinylated polyubiquitin,but with the biotinylated polyubiquitin at a concentration of lowermolarity than the target molar affinity constant for polyubiquitin. Thehigh affinity-binding phages can then be captured by streptavidin-coatedparamagnetic beads. Such “equilibrium capture” allows the antibodies tobe selected according to their affinities of binding, with sensitivitythat permits isolation of mutant clones with as little as two-foldhigher affinity from a great excess of phages with lower affinity.Conditions used in washing phages bound to a solid phase can also bemanipulated to discriminate on the basis of dissociation kinetics.

Anti-polyubiquitin clones may be activity selected. In one embodiment,the invention provides anti-polyubiquitin antibodies that block thebinding between a polyubiquitin ligand and polyubiquitin, but do notblock the binding between a polyubiquitin ligand and a second protein.Fv clones corresponding to such anti-polyubiquitin antibodies can beselected by (1) isolating anti-polyubiquitin clones from a phage libraryas described in Section B(I)(2) above, and optionally amplifying theisolated population of phage clones by growing up the population in asuitable bacterial host; (2) selecting polyubiquitin and a secondprotein against which blocking and non-blocking activity, respectively,is desired; (3) adsorbing the anti-polyubiquitin phage clones toimmobilized polyubiquitin; (4) using an excess of the second protein toelute any undesired clones that recognize polyubiquitin-bindingdeterminants which overlap or are shared with the binding determinantsof the second protein; and (5) eluting the clones which remain adsorbedfollowing step (4). Optionally, clones with the desiredblocking/non-blocking properties can be further enriched by repeatingthe selection procedures described herein one or more times.

DNA encoding the Fv clones of the invention is readily isolated andsequenced using conventional procedures (e.g. by using oligonucleotideprimers designed to specifically amplify the heavy and light chaincoding regions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of antibody-encoding DNAinclude Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) andPluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In one embodiment, a Fvclone derived from human variable DNA is fused to human constant regionDNA to form coding sequence(s) for all human, full or partial lengthheavy and/or light chains.

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

Other Methods of Generating Anti-Polyubiquitin Antibodies

Other methods of generating and assessing the affinity of antibodies arewell known in the art and are described, e.g., in Kohler et al., Nature256: 495 (1975); U.S. Pat. No. 4,816,567; Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986; Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987; Munson et al., Anal. Biochem., 107:220 (1980); Engels etal., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989); Abrahmsen et al.,EMBO J, 4: 3901 (1985); Methods in Enzymology, vol. 44 (1976); Morrisonet al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).

General Methods

In general, the invention provides affinity-matured anti-K63-linkedpolyubiquitin antibodies. These antibodies have increased affinity andspecificity for K63-linked polyubiquitin relative to the antibodiesdescribed in US Patent Publication No. US2007-0218069, incorporated byreference herein in its entirety. The best anti-K63-linked Fabidentified in that publication (Apu2.16) has a Kd of approximately 100nM and a small amount of artifactual binding to K48-linkedpolyubiquitin. The improved Fabs and antibodies provided herein areaffinity-matured versions of Apu2.16, and the best binders (see, forexample, the Fabs Apu3.A8, Apu3.A12, and Apu3.B3 and antibody versionsthereof) have a greater than ten-fold increase in affinity over Apu2.16for K63-linked polyubiquitin and negligible binding to K48-linkedpolyubiquitin. This increase in affinity and sensitivity permits themolecules of the invention to be used for applications and methods thatare benefited by (a) the increased sensitivity of the molecules of theinvention and/or (b) the tight binding of K63-linked polyubiquitin bythe molecules of the invention and/or (c) the increased specificity ofthe molecules of the invention for K63-linked polyubiquitin relative toK48-linked polyubiquitin, relative to the parental molecule Apu2.16.

In one embodiment, the invention provides anti-K63-linked polyubiquitinantibodies that are useful for treatment of K63-linkedpolyubiquitin-mediated disorders in which a partial or total blockade ofone or more K63-linked polyubiquitin activities is desired. In oneembodiment, the anti-K63-linked polyubiquitin antibodies of theinvention are used to treat cancer. In another embodiment, theanti-K63-linked polyubiquitin antibodies provided herein are used totreat muscular disorders, such as those indicated above. In anotherembodiment, the anti-K63-linked polyubiquitin antibodies provided hereinare used to treat neurological disorders, such as those indicated above.In another embodiment, the anti-K63-linked polyubiquitin antibodiesprovided herein are used to treat genetic disease. In anotherembodiment, the anti-K63-linked polyubiquitin antibodies provided hereinare used to treat immune/inflammatory disorders.

The unique properties of the anti-K63-linked polyubiquitin antibodies ofthe invention make them particularly useful for distinguishing betweendifferent lysine-linked forms of polyubiquitin in a cellular systemwithout resorting to cumbersome and expensive genetic manipulation orbiophysical methods such as mass spectrometry. The anti-K63-linkedpolyubiquitin antibodies of the invention can be used to characterizethe function(s) and activities of K63-linked polyubiquitins both invitro and in vivo. For example, as shown herein, both RIP and IRAK1 aremodulated in function and activity by alternative tagging with eitherK48-linked or K63-linked polyubiquitin due to TNFα or IL-1β stimulation,respectively. The anti-K63-linked polyubiquitin antibodies of theinvention permit the sensitive and specific detection of theK63-ubiquitinated versions of, e.g., RIP and/or IRAK in straightforwardand routine biomolecular assays such as immunoprecipitations, ELISAs, orimmunomicroscopy without the need for mass spectrometry or geneticmanipulation. In turn, this provides a significant advantage in bothobserving and elucidating the normal functioning of these pathways andin detecting when the pathways are functioning aberrantly.

The anti-K63-linked polyubiquitin antibodies of the invention can alsobe used to determine the role of specific lysine-linked polyubiquitinsin the development and pathogenesis of disease. For example, asdescribed above, the anti-K63-linked polyubiquitin antibodies of theinvention can be used to determine whether RIP or IRAK1 are aberrantlyK63-polyubiquitinated, which in turn provides information about thenormal or aberrant functioning of TNFα or IL-1β signaling, which can becorrelated with one or more disease states.

The anti-K63-linked polyubiquitin antibodies of the invention canfurther be used to treat diseases in which one or more K63-linkedpolyubiquitins are aberrantly regulated or aberrantly functioningwithout interfering with the normal activity of polyubiquitins for whichthe anti-polyubiquitin antibodies of the invention are not specific.

In another aspect, the anti-K63-linked polyubiquitin antibodies of theinvention find utility as reagents for detection and isolation ofK63-linked polyubiquitin, such as detection of K63-linked polyubiquitinin various cell types and tissues, including the determination ofK63-linked polyubiquitin density and distribution in cell populationsand within a given cell, and cell sorting based on the presence oramount of K63-linked polyubiquitin. The antibodies of the invention areable to specifically bind not only to isolated K63-linked polyubiquitinof various chain lengths, but also to proteins which have beenpolyubiquitinated with K63-linked polyubiquitin, and thus detection andbinding to either free (i.e. unconjugated to a heterologous protein) orK63-linked polyubiquitinated protein (i.e., conjugated to a heterologousprotein) and/or both is contemplated herein.

In yet another aspect, the present anti-K63-linked polyubiquitinantibodies are useful for the development of polyubiquitin antagonistswith blocking activity patterns similar to those of the subjectantibodies of the invention. For example, anti-K63-linked polyubiquitinantibodies of the invention can be used to determine and identify otherantibodies that have the same K63-linked polyubiquitin bindingcharacteristics and/or capabilities of blocking K63-linkedpolyubiquitin-mediated pathways.

As a further example, anti-K63-linked polyubiquitin antibodies of theinvention can be used to identify other anti-polyubiquitin antibodiesthat bind substantially the same antigenic determinant(s) of K63-linkedpolyubiquitin as the antibodies exemplified herein, including linear andconformational epitopes.

The anti-polyubiquitin antibodies of the invention can be used in assaysbased on the physiological pathways in which K63-linked polyubiquitin isinvolved to screen for small molecule antagonists of K63-linkedpolyubiquitin which will exhibit similar pharmacological effects inblocking the binding of one or more binding partners to K63-linkedpolyubiquitin as the antibody does. For example, K63-linkedpolyubiquitin is known to be involved in DNA repair (see, e.g., Pickartand Fushman, Curr. Opin. Chem. Biol. 8: 610-616 (2004)), and thus theactivity of anti-K63-linked polyubiquitin antibodies to antagonize a DNArepair pathway may be compared to the activity of one or more potentialsmall molecule antagonists of K63-linked polyubiquitin in that same DNArepair pathway. Similarly, in another example, K63-linked polyubiquitinis known to be involved in formation of Lewy bodies in Parkinson'sdisease (see, e.g., Lim et al., J. Neurosci. 25(8): 2002-9 (2005)), andthus the activity of anti-K63-linked polyubiquitin antibodies toantagonize the formation of Lewy bodies may be compared to the activityof one or more potential small molecule antagonists of K63-linkedpolyubiquitin in antagonizing the formation of Lewy bodies.

Generation of antibodies can be achieved using routine skills in theart, including those described herein, such as the hybridoma techniqueand screening of phage displayed libraries of binder molecules. Thesemethods are well-established in the art.

Briefly, the anti-polyubiquitin antibodies of the invention can be madeby using combinatorial libraries to screen for synthetic antibody cloneswith the desired activity or activities. In principle, syntheticantibody clones are selected by screening phage libraries containingphage that display various fragments of antibody variable region (Fv)fused to phage coat protein. Such phage libraries are panned by affinitychromatography against the desired antigen. Clones expressing Fvfragments capable of binding to the desired antigen are adsorbed to theantigen and thus separated from the non-binding clones in the library.The binding clones are then eluted from the antigen, and can be furtherenriched by additional cycles of antigen adsorption/elution. Any of theanti-polyubiquitin antibodies of the invention can be obtained bydesigning a suitable antigen screening procedure to select for the phageclone of interest followed by construction of a full lengthanti-polyubiquitin antibody clone using the Fv sequences from the phageclone of interest and suitable constant region (Fc) sequences describedin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. Seealso PCT Pub. WO03/102157, and references cited therein.

In one embodiment, anti-polyubiquitin antibodies of the invention aremonoclonal. Also encompassed within the scope of the invention areantibody fragments such as Fab, Fab′, Fab′-SH and F(ab′)₂ fragments, andvariations thereof, of the anti-polyubiquitin antibodies providedherein. These antibody fragments can be created by traditional means,such as enzymatic digestion, or may be generated by recombinanttechniques. Such antibody fragments may be chimeric, human or humanized.These fragments are useful for the experimental, diagnostic, andtherapeutic purposes set forth herein.

Monoclonal antibodies can be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The anti-polyubiquitin monoclonal antibodies of the invention can bemade using a variety of methods known in the art, including thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or alternatively they may be made by recombinant DNA methods(e.g., U.S. Pat. No. 4,816,567).

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Host cells include,but are not limited to, cells of either prokaryotic or eukaryotic(generally mammalian) origin. It will be appreciated that constantregions of any isotype can be used for this purpose, including IgG, IgM,IgA, IgD, and IgE constant regions, and that such constant regions canbe obtained from any human or animal species.

Generating Antibodies Using Prokaryotic Host Cells Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies of the invention. Such modulation isaccomplished at least in part by simultaneously modulating translationalstrengths for the polypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence. In certain embodiments, changes in the nucleotidesequence are silent. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

In one embodiment, a set of vectors is generated with a range of TIRstrengths for each cistron therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofthe desired antibody products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. Based on the translational strength comparison, the desiredindividual TIRs are selected to be combined in the expression vectorconstructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kan^(R) (U.S. Pat. No. 5,639,635). Other strains and derivativesthereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli _(λ)1776(ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, growth occurs at a temperature rangeincluding, but not limited to, about 20° C. to about 39° C., about 25°C. to about 37° C., and at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH can be from about 6.8 to about 7.4, orabout 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. In one embodiment, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, for example about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (a common carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized can be a column comprising a glass or silica surface, ora controlled pore glass column or a silicic acid column. In someapplications, the column is coated with a reagent, such as glycerol, topossibly prevent nonspecific adherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above can be applied onto a Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase would then be washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected generally is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II (e.g., primate metallothionein genes), adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene may first beidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR.Appropriate host cells when wild-type DHFR is employed include, forexample, the Chinese hamster ovary (CHO) cell line deficient in DHFRactivity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a polypeptide of interest (e.g., an antibody). Promotersequences are known for eukaryotes. Virtually all eukaryotic genes havean AT-rich region located approximately 25 to 30 bases upstream from thesite where transcription is initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellscan be controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, or from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding an antibody polypeptide of the inventionby higher eukaryotes can often be increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is generally located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (WI38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y.Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a humanhepatoma line (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are generally removed, forexample, by centrifugation or ultrafiltration. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a generally acceptable purification technique. The suitability ofaffinity reagents such as protein A as an affinity ligand depends on thespecies and isotype of any immunoglobulin Fc domain that is present inthe antibody. Protein A can be used to purify antibodies that are basedon human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to furtherpurification steps, as necessary, for example by low pH hydrophobicinteraction chromatography using an elution buffer at a pH between about2.5-4.5, generally performed at low salt concentrations (e.g., fromabout 0-0.25M salt).

It should be noted that, in general, techniques and methodologies forpreparing antibodies for use in research, testing and clinical use arewell-established in the art, consistent with the above and/or as deemedappropriate by one skilled in the art for the particular antibody ofinterest.

Activity Assays

Antibodies of the invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

Purified antibodies can be further characterized by a series of assaysincluding, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

Where necessary, antibodies are analyzed for their biological activity.In some embodiments, antibodies of the invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays.

In one embodiment, the invention contemplates an altered antibody thatpossesses some but not all effector functions, which make it a desirablecandidate for many applications in which the half life of the antibodyin vivo is important yet certain effector functions (such as complementand ADCC) are unnecessary or deleterious. In certain embodiments, the Fcactivities of the antibody are measured to ensure that only the desiredproperties are maintained. In vitro and/or in vivo cytotoxicity assayscan be conducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express FcγRIII only, whereasmonocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitroassay to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and Natural Killer (NK) cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., in aanimal model such as that disclosed in Clynes et al. PNAS (USA)95:652-656 (1998). C1q binding assays may also be carried out to confirmthat the antibody is unable to bind C1q and hence lacks CDC activity. Toassess complement activation, a CDC assay, e.g. as described inGazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may beperformed. FcRn binding and in vivo clearance/half life determinationscan also be performed using methods known in the art.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human Antibodies

Human anti-polyubiquitin antibodies of the invention can be constructedby combining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s) as described above. Alternatively, human monoclonalanti-polyubiquitin antibodies of the invention can be made by thehybridoma method. Human myeloma and mouse-human heteromyeloma cell linesfor the production of human monoclonal antibodies have been described,for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is forpolyubiquitin including a specific lysine linkage and the other is forany other antigen. In certain embodiments, bispecific antibodies maybind to two different polyubiquitins having two different lysinelinkages. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments (e.g. F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J, 10: 3655 (1991).

According to a different embodiment, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The dimerization domain comprises (or consists of), forexample, an Fc region or a hinge region. In this scenario, the antibodywill comprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. In one embodiment, a multivalentantibody comprises (or consists of), for example, three to about eight,or four antigen binding sites. The multivalent antibody comprises atleast one polypeptide chain (for example, two polypeptide chains),wherein the polypeptide chain(s) comprise two or more variable domains.For instance, the polypeptide chain(s) may compriseVD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is asecond variable domain, Fc is one polypeptide chain of an Fc region, X1and X2 represent an amino acid or polypeptide, and n is 0 or 1. Forinstance, the polypeptide chain(s) may comprise: VH—CH1-flexiblelinker-VH—CH1-Fc region chain; or VH—CH1-VH—CH1-Fc region chain. Themultivalent antibody herein may further comprise at least two (forexample, four) light chain variable domain polypeptides. The multivalentantibody herein may, for instance, comprise from about two to abouteight light chain variable domain polypeptides. The light chain variabledomain polypeptides contemplated here comprise a light chain variabledomain and, optionally, further comprise a CL domain. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics. The amino acidalterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table A under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table A,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE A Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (O)(3) acidic: Asp (D), Glu (E)(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibodies thusgenerated are displayed from filamentous phage particles as fusions toat least part of a phage coat protein (e.g., the gene III product ofM13) packaged within each particle. The phage-displayed variants arethen screened for their biological activity (e.g. binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, scanning mutagenesis (e.g., alanine scanning)can be performed to identify hypervariable region residues contributingsignificantly to antigen binding. Alternatively, or additionally, it maybe beneficial to analyze a crystal structure of the antigen-antibodycomplex to identify contact points between the antibody and antigen.Such contact residues and neighboring residues are candidates forsubstitution according to techniques known in the art, including thoseelaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening using techniques known in the art,including those described herein, and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants.

In one aspect, the invention provides antibodies comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

Immunoconjugates

In another aspect, the invention provides immunoconjugates, orantibody-drug conjugates (ADC), comprising an antibody conjugated to acytotoxic agent such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubutin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is tested for the treatment of cancers that express CanAg,such as colon, pancreatic, gastric, and others. MLN-2704 (MillenniumPharm., BZL Biologics, Immunogen Inc.), an antibody drug conjugatecomposed of the anti-prostate specific membrane antigen (PSMA)monoclonal antibody linked to the maytansinoid drug moiety, DM1, istested for the potential treatment of prostate tumors. The auristatinpeptides, auristatin E (AE) and monomethylauristatin (MMAE), syntheticanalogs of dolastatin, were conjugated to chimeric monoclonal antibodiescBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 onhematological malignancies) (Doronina et al (2003) Nature Biotechnology21(7):778-784) and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (above). Enzymatically active toxins and fragmentsthereof that can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis-(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolostatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Exemplary embodiments of maytansinoid drug moieties include: DM1; DM3;and DM4. Immunoconjugates containing maytansinoids, methods of makingsame, and their therapeutic use are disclosed, for example, in U.S. Pat.Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates can be prepared by chemically linkingan antibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Maytansinoids include, but are not limited to,maytansinol and maytansinol analogues modified in the aromatic ring orat other positions of the maytansinol molecule, such as variousmaytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 BI, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents. Additional linkinggroups are described and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis-(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Coupling agents include, but are not limited to,N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al.,Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In one embodiment, the linkage isformed at the C-3 position of maytansinol or a maytansinol analogue.

Auristatins and Dolostatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. 5663149) and antifungal activity (Pettit et al(1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin orauristatin drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Exemplary auristatin embodiments include MMAE and MMAF. Additionalexemplary embodiments comprising MMAE or MMAF and various linkercomponents (described further herein) include Ab-MC-vc-PAB-MMAF,Ab-MC-vc-PAB-MMAE, Ab-MC-MMAE and Ab-MC-MMAF.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics is capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I)₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug to which the antibody canbe conjugated is QFA, which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampleTc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, MRI), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis-(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing ADC are described herein.

Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC’), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzyme, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Exemplary linker component structures are shown below (wherein the wavyline indicates sites of covalent attachment to other components of theADC):

Additional exemplary linker components and abbreviations include(wherein the antibody (Ab) and linker are depicted, and p is 1 to about8):

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either galactose oxidase or sodium meta-periodate mayyield carbonyl (aldehyde and ketone) groups in the protein that canreact with appropriate groups on the drug (Hermanson, BioconjugateTechniques). In another embodiment, proteins containing N-terminalserine or threonine residues can react with sodium meta-periodate,resulting in production of an aldehyde in place of the first amino acid(Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. 5362852).Such aldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent to one another or separated bya region encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Antibody (Ab)-MC-MMAE may be prepared by conjugation of any of theantibodies provided herein with MC-MMAE as follows. Antibody, dissolvedin 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treatedwith an excess of 100 mM dithiothreitol (DTT). After incubation at 37°C. for about 30 minutes, the buffer is exchanged by elution overSephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol/Abvalue is checked by determining the reduced antibody concentration fromthe absorbance at 280 nm of the solution and the thiol concentration byreaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of theabsorbance at 412 nm. The reduced antibody dissolved in PBS is chilledon ice. The drug linker reagent, maleimidocaproyl-monomethyl auristatinE (MMAE), i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrileand water at known concentration, and added to the chilled reducedantibody 2H9 in PBS. After about one hour, an excess of maleimide isadded to quench the reaction and cap any unreacted antibody thiolgroups. The reaction mixture is concentrated by centrifugalultrafiltration and 2H9-MC-MMAE is purified and desalted by elutionthrough G25 resin in PBS, filtered through 0.2 μm filters under sterileconditions, and frozen for storage.

Antibody-MC-MMAF may be prepared by conjugation of any of the antibodiesprovided herein with MC-MMAF following the protocol provided forpreparation of Ab-MC-MMAE.

Antibody-MC-val-cit-PAB-MMAE is prepared by conjugation of any of theantibodies provided herein with MC-val-cit-PAB-MMAE following theprotocol provided for preparation of Ab-MC-MMAE.

Antibody-MC-val-cit-PAB-MMAF is prepared by conjugation of any of theantibodies provided herein with MC-val-cit-PAB-MMAF following theprotocol provided for preparation of Ab-MC-MMAE.

Antibody-SMCC-DM1 is prepared by conjugation of any of the antibodiesprovided herein with SMCC-DM1 as follows. Purified antibody isderivatized with (Succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, PierceBiotechnology, Inc) to introduce the SMCC linker. Specifically, antibodyis treated at 20 mg/mL in 50 mM potassium phosphate/50 mM sodiumchloride/2 mM EDTA, pH 6.5 with 7.5 molar equivalents of SMCC (20 mM inDMSO, 6.7 mg/mL). After stirring for 2 hours under argon at ambienttemperature, the reaction mixture is filtered through a Sephadex G25column equilibrated with 50 mM potassium phosphate/50 mM sodiumchloride/2 mM EDTA, pH 6.5. Antibody-containing fractions are pooled andassayed.

Antibody-SMCC prepared thusly is diluted with 50 mM potassiumphosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5, to a finalconcentration of about 10 mg/ml, and reacted with a 10 mM solution ofDM1 in dimethylacetamide. The reaction is stirred at ambient temperatureunder argon for 16.5 hours. The conjugation reaction mixture is filteredthrough a Sephadex G25 gel filtration column (1.5×4.9 cm) with 1×PBS atpH 6.5. The DM1 drug to antibody ratio (p) may be about 2 to 5, asmeasured by the absorbance at 252 nm and at 280 nm.

Ab-SPP-DM1 is prepared by conjugation of any of the antibodies providedherein with SPP-DM1 as follows. Purified antibody is derivatized withN-succinimidyl-4-(2-pyridylthio)pentanoate to introduce dithiopyridylgroups. Antibody (376.0 mg, 8 mg/mL) in 44.7 mL of 50 mM potassiumphosphate buffer (pH 6.5) containing NaCl (50 mM) and EDTA (1 mM) istreated with SPP (5.3 molar equivalents in 2.3 mL ethanol). Afterincubation for 90 minutes under argon at ambient temperature, thereaction mixture is gel filtered through a Sephadex G25 columnequilibrated with a 35 mM sodium citrate, 154 mM NaCl, 2 mM EDTA buffer.Antibody-containing fractions were pooled and assayed. The degree ofmodification of the antibody is determined as described above.

Antibody-SPP-Py (about 10 μmoles of releasable 2-thiopyridine groups) isdiluted with the above 35 mM sodium citrate buffer, pH 6.5, to a finalconcentration of about 2.5 mg/mL. DM1 (1.7 equivalents, 17 μmoles) in3.0 mM dimethylacetamide (DMA, 3% v/v in the final reaction mixture) isthen added to the antibody solution. The reaction proceeds at ambienttemperature under argon for about 20 hours. The reaction is loaded on aSephacryl S300 gel filtration column (5.0 cm×90.0 cm, 1.77 L)equilibrated with 35 mM sodium citrate, 154 mM NaCl, pH 6.5. The flowrate may be about 5.0 mL/min and 65 fractions (20.0 mL each) arecollected. The number of DM1 drug molecules linked per antibody molecule(p′) is determined by measuring the absorbance at 252 nm and 280 nm, andmay be about 2 to 4 DM1 drug moieties per 2H9 antibody.

Antibody-BMPEO-DM1 is prepared by conjugation of any of the antibodiesprovided herein with BMPEO-DM1 as follows. The antibody is modified bythe bis-maleimido reagent BM(PEO)₄ (Pierce Chemical), leaving anunreacted maleimido group on the surface of the antibody. This may beaccomplished by dissolving BM(PEO)₄ in a 50% ethanol/water mixture to aconcentration of 10 mM and adding a tenfold molar excess to a solutioncontaining antibody in phosphate buffered saline at a concentration ofapproximately 1.6 mg/ml (10 micromolar) and allowing it to react for 1hour to form an antibody-linker intermediate, 2H9-BMPEO. Excess BM(PEO)₄is removed by gel filtration (HiTrap column, Pharmacia) in 30 mMcitrate, pH 6 with 150 mM NaCl buffer. An approximate 10 fold molarexcess DM1 is dissolved in dimethyl acetamide (DMA) and added to the2H9-BMPEO intermediate. Dimethyl formamide (DMF) may also be employed todissolve the drug moiety reagent. The reaction mixture is allowed toreact overnight before gel filtration or dialysis into PBS to removeunreacted DM1. Gel filtration on S200 columns in PBS is used to removehigh molecular weight aggregates and to furnish purified 2H9-BMPEO-DM1.

Antibody Derivatives

Antibodies of the invention can be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. In one embodiment, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, the polymers can be the sameor different molecules. In general, the number and/or type of polymersused for derivatization can be determined based on considerationsincluding, but not limited to, the particular properties or functions ofthe antibody to be improved, whether the antibody derivative will beused in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)).The radiation may be of any wavelength, and includes, but is not limitedto, wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theantibody-nonproteinaceous moiety are killed.

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, including, butnot limited to those with complementary activities that do not adverselyaffect each other. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

An antibody of the invention may be used in, for example, in vitro, exvivo and in vivo therapeutic methods. Antibodies of the invention can beused as an antagonist to partially or fully block the specific antigenactivity in vitro, ex vivo and/or in vivo. Moreover, at least some ofthe antibodies of the invention can neutralize antigen activity fromother species. Accordingly, antibodies of the invention can be used toinhibit a specific antigen activity, e.g., in a cell culture containingthe antigen, in human subjects or in other mammalian subjects having theantigen with which an antibody of the invention cross-reacts (e.g.chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). Inone embodiment, an antibody of the invention can be used for inhibitingantigen activities by contacting the antibody with the antigen such thatantigen activity is inhibited. In one embodiment, the antigen is a humanprotein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor inhibiting an antigen in a subject suffering from a disorder inwhich the antigen activity is detrimental, comprising administering tothe subject an antibody of the invention such that the antigen activityin the subject is inhibited. In one embodiment, the antigen is a humanprotein molecule and the subject is a human subject. Alternatively, thesubject can be a mammal expressing the antigen with which an antibody ofthe invention binds. Still further the subject can be a mammal intowhich the antigen has been introduced (e.g., by administration of theantigen or by expression of an antigen transgene). An antibody of theinvention can be administered to a human subject for therapeuticpurposes. Moreover, an antibody of the invention can be administered toa non-human mammal expressing an antigen with which the antibodycross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). Antibodies of the invention can be used to treat,inhibit, delay progression of, prevent/delay recurrence of, ameliorate,or prevent diseases, disorders or conditions associated with abnormalexpression and/or activity of polyubiquitins and polyubiquitinatedproteins, including but not limited to cancer, muscular disorders,ubiquitin-pathway-related genetic disorders, immune/inflammatorydisorders, neurological disorders, and other ubiquitin pathway-relateddisorders.

In one aspect, a blocking antibody of the invention is specific for apolyubiquitin having a K63 lysine linkage, and inhibits normalK63-linked polyubiquitin activity by blocking or interfering with theinteraction between a K63-linked polyubiquitin and a protein thatinteracts with that K63-linked polyubiquitin, thereby inhibiting thecorresponding signal pathway and other associated molecular or cellularevents. In another aspect, a blocking antibody of the invention specificfor K63-linked polyubiquitin interacts with one or more proteinsconjugated with K63-linked polyubiquitin, thereby inhibiting theinteraction of the protein with signal pathways or other bindingpartners and interfering with associated molecular or cellular events.

In certain embodiments, an immunoconjugate comprising an antibody of theinvention conjugated with a cytotoxic agent is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by cells expressing one or moreproteins on their cell surface which are associated with K63-linkedpolyubiquitin, resulting in increased therapeutic efficacy of theimmunoconjugate in killing the target cell with which it is associated.In one embodiment, the cytotoxic agent targets or interferes withnucleic acid in the target cell. Examples of such cytotoxic agentsinclude any of the chemotherapeutic agents noted herein (such as amaytansinoid or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with another antibody, and/oradjuvant/therapeutic agents (e.g., steroids). For instance, an antibodyof the invention may be combined with an anti-inflammatory and/orantiseptic in a treatment scheme, e.g. in treating any of the diseasesdescribed herein, including cancer, muscular disorders,ubiquitin-pathway-related genetic disorders, immune/inflammatorydisorders, neurological disorders, and other ubiquitin pathway-relateddisorders. Such combined therapies noted above include combinedadministration (where the two or more agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,and/or following, administration of the adjunct therapy or therapies.

An antibody of the invention (and adjunct therapeutic agent) can beadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, e.g. by injections, suchas intravenous or subcutaneous injections, depending in part on whetherthe administration is brief or chronic.

The location of the binding target of an antibody of the invention maybe taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described in Marasco, Gene Therapy 4: 11-15(1997); Kontermann, Methods 34: 163-170 (2004); U.S. Pat. Nos. 6,004,940and 6,329,173; U.S. Patent Application Publication No. 2003/0104402, andPCT Publication No. WO2003/077945. Intracellular expression of anintrabody is effected by introducing a nucleic acid encoding the desiredantibody or antigen-binding portion thereof (lacking the wild-typeleader sequence and secretory signals normally associated with the geneencoding that antibody or antigen-binding fragment) into a target cell.Any standard method of introducing nucleic acids into a cell may beused, including, but not limited to, microinjection, ballisticinjection, electroporation, calcium phosphate precipitation, liposomes,and transfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest. One or morenucleic acids encoding all or a portion of an anti-polyubiquitinantibody of the invention can be delivered to a target cell, such thatone or more intrabodies are expressed which are capable of intracellularbinding to a polyubiquitin and modulation of one or morepolyubiquitin-mediated cellular pathways.

In another embodiment, internalizing antibodies are provided. Antibodiescan possess certain characteristics that enhance delivery of antibodiesinto cells, or can be modified to possess such characteristics.Techniques for achieving this are known in the art. For example,cationization of an antibody is known to facilitate its uptake intocells (see, e.g., U.S. Pat. No. 6,703,019). Lipofections or liposomescan also be used to deliver the antibody into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the binding domain of the target protein is generallyadvantageous. For example, based upon the variable-region sequences ofan antibody, peptide molecules can be designed that retain the abilityto bind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

Entry of modulator polypeptides into target cells can be enhanced bymethods known in the art. For example, certain sequences, such as thosederived from HIV Tat or the Antennapedia homeodomain protein are able todirect efficient uptake of heterologous proteins across cell membranes.See, e.g., Chen et al., Proc. Natl. Acad. Sci. USA (1999), 96:4325-4329.

When the binding target is located in the brain, certain embodiments ofthe invention provide for the antibody or antigen-binding fragmentthereof to traverse the blood-brain barrier. Certain neurodegenerativediseases are associated with an increase in permeability of theblood-brain barrier, such that the antibody or antigen-binding fragmentcan be readily introduced to the brain. When the blood-brain barrierremains intact, several art-known approaches exist for transportingmolecules across it, including, but not limited to, physical methods,lipid-based methods, and receptor and channel-based methods.

Physical methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,circumventing the blood-brain barrier entirely, or by creating openingsin the blood-brain barrier. Circumvention methods include, but are notlimited to, direct injection into the brain (see, e.g., Papanastassiouet al., Gene Therapy 9: 398-406 (2002)), interstitialinfusion/convection-enhanced delivery (see, e.g., Bobo et al., Proc.Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and implanting a deliverydevice in the brain (see, e.g., Gill et al., Nature Med. 9: 589-595(2003); and Gliadel Wafers™, Guildford Pharmaceutical). Methods ofcreating openings in the barrier include, but are not limited to,ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086),osmotic pressure (e.g., by administration of hypertonic mannitol(Neuwelt, E. A., Implication of the Blood-Brain Barrier and itsManipulation, Vols 1 & 2, Plenum Press, N.Y. (1989))), permeabilizationby, e.g., bradykinin or permeabilizer A-7 (see, e.g., U.S. Pat. Nos.5,112,596, 5,268,164, 5,506,206, and 5,686,416), and transfection ofneurons that straddle the blood-brain barrier with vectors containinggenes encoding the antibody or antigen-binding fragment (see, e.g., U.S.Patent Publication No. 2003/0083299).

Lipid-based methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,encapsulating the antibody or antigen-binding fragment in liposomes thatare coupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody orantigen-binding fragment in low-density lipoprotein particles (see,e.g., U.S. Patent Application Publication No. 20040204354) orapolipoprotein E (see, e.g., U.S. Patent Application Publication No.20040131692).

Receptor and channel-based methods of transporting the antibody orantigen-binding fragment across the blood-brain barrier include, but arenot limited to, using glucocorticoid blockers to increase permeabilityof the blood-brain barrier (see, e.g., U.S. Patent ApplicationPublication Nos. 2002/0065259, 2003/0162695, and 2005/0124533);activating potassium channels (see, e.g., U.S. Patent ApplicationPublication No. 2005/0089473), inhibiting ABC drug transporters (see,e.g., U.S. Patent Application Publication No. 2003/0073713); coatingantibodies with a transferrin and modulating activity of the one or moretransferrin receptors (see, e.g., U.S. Patent Application PublicationNo. 2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat.No. 5,004,697).

The antibody composition of the invention would be formulated, dosed,and administered in a fashion consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of the disorder,the site of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The antibody need not be, but is optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question. The effective amount of such other agents depends on theamount of antibodies of the invention present in the formulation, thetype of disorder or treatment, and other factors discussed above. Theseare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with otheragents such as chemotherapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition effectivefor treating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice. Moreover,the article of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises anantibody of the invention; and (b) a second container with a compositioncontained therein, wherein the composition comprises a further cytotoxicor otherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES Example 1 Affinity Maturation of a K63-LinkedDiubiquitin-Specific Antibody Fragment

Panels of antibodies able to discriminate between K48-linkedpolyubiquitin and K63-linked polyubiquitin were described in US PatentPublication No. US2007-0218069, incorporated by reference herein in itsentirety. The affinity and specificity of the best anti-K63-linked Fabidentified in that publication (Apu2.16, Kd of approximately 100 nM anda small amount of artifactual binding to K48-linked polyubiquitin) wasinferior to the affinity and specificity of the best anti-K48-linked Fabidentified in that publication (Apu2.07, Kd of approximately 1 nM, noobserved binding to K63-linked polyubiquitin). An improved K63-linkedpolyubiquitin-specific Fab/antibody was sought to facilitateapplications in which greater specificity or affinity of theFab/antibody would be desirable.

(A) Library Generation

The Apu2.16 anti-K63-linked diubiquitin antibody fragment (Fab) wassubjected to mutagenesis in order to affinity mature the antibody.Residues 49, 50, 52, and 53 of CDR L2 and residues 50, 52, 53, 54, and56 of CDR H2 were selected for mutagenesis based on their contact withK63-linked diubiquitin, as shown in the co-crystal structure of Apu2.16and K63-linked diubiquitin (see FIG. 2 and US Patent Publication No.US2007-0218069, incorporated herein in its entirety). An Apu2.16 stoptemplate with TAA stop codons at positions 51 of CDR L2 and 52a of CDRH2 was used to force mutagenesis of both CDR regions. Expression of thisconstruct was under the control of the bacterial alkaline phosphatase(PhoA) promoter. Both the light chain and the heavy chain contained anamino terminal bacterial stII signal sequence to permit secretion in E.coli. The heavy chain carboxyl terminus was fused in-frame to an amberstop codon followed by gene product III of the M13 bacteriophage,allowing for monovalent Fab display on phage when expressed in an ambersuppressor E. coli strain. Degenerate oligonucleotides were synthesizedto soft-randomize the positions of contact so that 50% of the time theApu2.16 wild-type residue would be retained and 50% of the time one ofthe remaining 19 amino acids would be encoded. To achieve softrandomization, oligonucleotides were designed such that certainnucleotide positions were occupied 70% of the time with the indicatedbase and 10% of the time occupied by one of the three other bases(Gallop et al., J. Med. Chem. 37: 1233 (1994)). Where such softrandomization was included at a particular base, the presence of thesoft randomization is indicated by the presence of a number at that baseposition. The number “5” indicates that the base adenine was present 70%of the time at that position, while the bases guanine, cytosine, andthymine were each present 10% of the time. Similarly, the number “6”refers to guanine, “7” to cytosine, and “8” to thymine, where in eachcase, each of the other three bases was present only 10% of the time.

Mutagenic oligonucleotides 310887(CCGAAGCTTCTGATT857876GCA877567CTCTACTCTGGAGTC) (SEQ ID NO: 1) and310890 (GGCCTGGAATGGGTTGCA858ATT878CCT858858GGC 878ACTTCTTATGCCGATAGC)(SEQ ID NO: 2) were used with 40 μg of Kunkel DNA of the Apu2.16 stoptemplate in a Kunkel mutagenesis reaction (see Kunkel, Proc. Natl. Acad.Sci. USA 82: 488 (1985) and Sidhu et al., Meth. Enzymol. 328: 333(2000)). The mutagenesis reaction was electroporated into ElectroTenBlue E. coli (Stratagene) and recovered in 25 mL of SOC medium for 45minutes at 37° C. with shaking. Twenty microliters were removed andten-fold serial dilutions were plated onto solid agar plates containingcarbenicillin and grown overnight at 37° C. to determine the librarysize. The remaining culture was transferred to 500 mL of 2YT brothcontaining 50 μg/mL carbenicillin, 50 μg/mL kanamycin, and 1010 phage/mLM13K07 helper phage (New England Biolabs). The culture was grown for 14hours at 30° C. with shaking. The library contained approximately 3×10¹⁰CFUs. The phage were purified from the culture supernatant by two roundsof precipitation with ⅕ volume of 20% polyethylene glycol (PEG)/2.5 MNaCl.

(B) Library Sorting

Amplified phage were used in sorting against enzymatically synthesizedK63-linked diubiquitin (Boston Biochem) immobilized on 96-well Maxisorbimmunoplates (Nunc). The plates were coated overnight at 4° C. with 5μg/mL K63-linked diubiquitin in 50 mM sodium carbonate buffer, pH 9.6.The coated plates were blocked with 2.5% milk in PBS containing 0.05%Tween-20 (PBST) for one hour at 25° C. with shaking. The phage werediluted to an OD₂₆₈ of 5.0 in 2.5% milk/PBST and incubated on ice forone hour. After blocking, the plate was washed five times with PBST. Onehundred microliters/well of the phage was added and incubated at 25° C.for one hour with shaking. After binding, the plate was washed ten timeswith PBST. Phage were eluted with 100 μL/well of 100 mM HCl for 20minutes at 25° C. with shaking. The eluate was neutralized with 1/10volume 1 M Tris, pH 11.0 and subsequently propagated in XL-1 blue E.coli (Stratagene) with the addition of M13K07 helper phage.

Amplified phage were used in subsequent rounds of sorting. The secondsort was performed as described above, with the exception that 100 nMbiotinylated K63-linked diubiquitin was used as a target in solution andthe phage were used at an OD₂₆₈ of 1.0. The biotinylated K63-linkeddiubiquitin along with bound phage were captured on 5 μg/mLneutravidin-coated immunoplates. The third sorting round was performedsimilarly to the second round, with the addition of soluble 100 nMK48-linked diubiquitin and 100 nM monoubiquitin (Boston Biochem) ascompetitors in the phage blocking and phage binding steps. In the fourthsorting round the biotinylated K63-linked diubiquitin targetconcentration was decreased to 10 nM and the K48-linked diubiquitin andmonoubiquitin competitor concentrations were increased to 1 μM each.Enrichment for binding to biotinylated K63-linked diubiquitin comparedto neutravidin alone was observed after rounds three and four.

Ninety-six individual clones from the fourth sort were grown up in a96-well format in 1 mL of 2YT broth containing 50 μg/mL carbenicillinand 1010 phage/mL M13K07 helper phage. Supernatants from those cultureswere used in high-throughput phage ELISAs for binding to K63-linkeddiubiquitin, K48-linked diubiquitin, monoubiquitin, or the uncoatedwells of the plate. Fifty-one clones demonstrated specific binding toK63-linked diubiquitin and their DNA was sequenced using standardprocedures. The sequences of CDRs L2 and H2 for each clone are shown inTable B. CDRs L1, L3, H1, and H3 were not sequenced, but since they werenot targeted for mutagenesis they were expected to retain the Apu2.16template CDR L1 sequence (RASQSVSSAVA) (SEQ ID NO: 3), CDR L3 sequence(QQYSSYSSLFT) (SEQ ID NO: 4), CDR H1 sequence (VKTGLI) (SEQ ID NO: 5),and CDR H3 sequence (EYYRWYTAI) (SEQ ID NO: 6), which had not beentargeted for mutagenesis.

TABLE B HVR L2 and HVR H2 Sequences for Parental and Affinity-MaturedAnti-K63-linked Polyubiquitin Fabs SEQ SEQ HVR L2 sequence ID HVR H2sequence ID Clone 49 50 51 52 53 54 55 56 NO 50 51 52 52a 53 54 55 56 5758 59 60 61 62 63 64 65 NO Apu2.16 Y S A S S L Y S 7 Y I S P Y Y G S T SY A D S V K G 59 A8 Y S A R S L Y S 8 Y I T P Y Y G S T S Y A D S V K G60 A9 Y S A V S L Y S 9 Y I S P Y Y G S T S Y A D S V K G 61 A11 Y S A SS L Y S 10 D I T P Y Y G S T S Y A D S V K G 62 A12 Y S A R S L Y S 11 YI A P Y Y G S T S Y A D S V K G 63 B1 Y S A R S L Y S 12 Y I T P Y Y G ST S Y A D S V K G 64 B2 Y S A R S L Y S 13 Y I T P Y Y G S T S Y A D S VK G 65 B3 Y A A A S L Y S 14 Y I F P Y H G S T S Y A D S V K G 66 B4 Y SA V S L Y S 15 D I A P Y Y G S T S Y A D S V K G 67 B5 Y S A A S L Y S16 Y I A P Y Y G A T S Y A D S V K G 68 B9 Y S A R S L Y S 17 Y I F P YF G S T S Y A D S V K G 69 C1 Y A A A S L Y S 18 Y I A P Y Y G S T S Y AD S V K G 70 C3 Y S A V S L Y S 19 D I A P Y Y G S T S Y A D S V K G 71C4 Y S A V S L Y S 20 Y I Y P Y Y G S T S Y A D S V K G 72 C5 Y S A R SL Y S 21 Y I S P Y Y G S T S Y A D S V K G 73 C8 Y S A R S L Y S 22 Y IT P Y Y G S T S Y A D S V K G 74 C12 Y A A A S L Y S 23 Y I T P Y Y G ST S Y A D S V K G 75 D1 Y A A V S L Y S 24 Y I S P Y Y G W T S Y A D S VK G 76 D3 Y S A T S L Y S 25 D I S P Y Y G S T S Y A D S V K G 77 D7 Y SA N S L Y S 26 Y I A P Y Y G S T S Y A D S V K G 78 D8 Y A A A S L Y S27 Y I V P Y Y G S T S Y A D S V K G 79 D9 Y S A V S L Y S 28 D I F P YY G S S S Y A D S V K G 80 D10 Y S A S S L Y S 29 D I V P Y Y G S T S YA D S V K G 81 D11 Y S A V S L Y S 30 Y I F P Y Y G S T S Y A D S V K G82 E1 Y S A V S L Y S 31 D I S P Y Y G S T S Y A D S V K G 83 E2 F A A AS L Y S 32 Y I A P Y Y G S T S Y A D S V K G 84 E3 Y S A R S L Y S 33 YI A P Y Y G S T S Y A D S V K G 85 E4 Y S A S S L Y S 34 W I S P Y Y G ST S Y A D S V K G 86 E5 Y S A A S L Y S 35 Y I A P Y Y G S T S Y A D S VK G 87 E8 Y S A A S L Y S 36 Y I T P Y Y G S T S Y A D S V K G 88 E12 YA A R S L Y S 37 D I T P Y F G F T S Y A D S V K G 89 F1 Y S A V S L Y S38 Y I S P Y L G S T S Y A D S V K G 90 F3 Y A A V S L Y S 39 Y I F P YY G C T S Y A D S V K G 91 F4 Y S A L S L Y S 40 Y I Y P Y Y G W T S Y AD S V K G 92 F6 Y S A A S L Y S 41 D I S P Y Y G S T S Y A D S V K G 93F9 Y S A S S L Y S 42 D I T P Y Y G F T S Y A D S V K G 94 G1 Y S A S SL Y S 43 Y I A P Y Y G S T S Y A D S V K G 95 G2 Y S A S S L Y S 44 D IS P Y Y G S T S Y A D S V K G 96 G3 Y S A A S L Y S 45 D I S P Y Y G S TS Y A D S V K G 97 G4 Y S A T S L Y S 46 Y I T P Y Y G S T S Y A D S V KG 98 G5 Y S A L S L Y S 47 Y I A P Y Y G S T S Y A D S V K G 99 G10 Y SA V S L Y S 48 D I T P Y Y G F T S Y A D S V K G 100 G11 Y A A A S L Y S49 D I T P Y Y G S T S Y A D S V K G 101 G12 Y S A S S L Y S 50 D I T PY Y G S T S Y A D S V K G 102 H2 Y A A A S L Y S 51 D I T P Y Y G S T SY A D S V K G 103 H3 Y A A A S L Y S 52 Y I T P Y Y G S T S Y A D S V KG 104 H4 Y S A A S L Y S 53 Y I S P Y Y G S T S Y A D S V K G 105 H5 Y AA A S L Y S 54 D I S P Y Y G S T S Y A D S V K G 106 H6 Y S A S S L Y S55 Y I T P Y Y G F T S Y A D S V K G 107 H7 Y S A V S L Y S 56 Y I T P YY G S T S Y A D S V K G 108 H9 Y S A V S L Y S 57 D I T P Y Y G F T S YA D S V K G 109 H10 Y S A L S L Y S 58 Y I A P Y Y G S T S Y A D S V K G110 Con- Y/F A/S A S/R/ S L Y S 111 Y/D/ I S/T/ P Y Y/F/ G S/G T S Y A DS V K G 112 sensus V/T/ W A/F/ L/H A/F A/N/ Y/V W L

(C) Fab Production

Twenty-four of the most specific anti-K63-linked diubiquitin clones (asjudged by a signal ratio of greater than ten for binding to K63-linkeddiubiquitin relative to K48-linked diubiquitin in the phage spot ELISAdescribed above) were selected for soluble Fab production. Plasmidsencoding these Fabs were transformed into E. coli and plated on solidagar containing carbenicillin. Single colonies were used to inoculate 25mL of 2YT broth containing 50 μg/mL carbenicillin. Cultures were grownovernight at 37° C. and 5 mL were used to inoculate 500 mL of completeC.R.A.P. media (3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate 2H₂O, 1.07 gKCl, 5.36 g yeast extract, 5.36 g Hycase SF (Sheffield), pH adjusted to7.3 by addition of KOH and volume adjusted to 872 mL with ultrapurewater, autoclaved, cooled to 55° C., to which was added (per L) 110 mL 1M MOPS pH 7.3, 11 mL 50% glucose, and 7 mL 1 M MgSO₄) with 50 μg/mLcarbenicillin. Cultures were grown at 30° C. for 24 hours with shaking.Cells were harvested by centrifugation and pellets were stored at −20°C. Fabs were purified by resuspending each pellet in 35 mL cold washbuffer (PBS+150 mM NaCl) containing 10 μg/mL DNase 1, 0.2 mg/mLlysozyme, and 1 mM phenylmethylsulphonylfluoride (PMSF). Pellets wereresuspended by vortexing rapidly for 45 minutes at 25° C. Cell debriswas pelleted by centrifugation and lysates were loaded on 1 mL proteinA-sepharose columns (GE Health Sciences) preequilibrated with cold washbuffer. The columns were washed with 50 mL cold wash buffer, eluted with3 mL 0.1 M acetic acid, and neutralized with 150 μL of 2M Tris, pH 11.0.The Fabs were concentrated using Amicon Ultra-15 centrifugal filterunits (5 KD cut-off, Millipore). The resulting Fab concentrations weredetermined spectrophotometrically, and the concentrated Fabs were storedat 4° C.

(D) Affinity Analysis of Isolated Fabs

The affinities of the 24 selected Fabs (see Example 1(C), above), weredetermined by surface plasmon resonance (SPR) using a BIACORE™ A100system (Biacore). Approximately 50 resonance units of K63-linkeddiubiquitin or K48-linked diubiquitin were immobilized in two of thefour flow cells of a CM5 chip using the amine coupling protocol suppliedby the manufacturer. One flow cell on each CM5 chip was activated andethanolamine blocked without immobilizing protein, to be used as areference. Two-fold serial dilutions (31.25-500 nM) of each Fab wereinjected (60 μL total at a flow rate of 30 μL/min) over each flow cell.The reference signal was subtracted from each flow cell signal.Following a dissociation period (10 minutes), the chip surface wasregenerated with 15 μL of 10 mM HCl. Kinetic constants and bindingconstants were simultaneously calculated by nonlinear regressionanalysis, using software provided by the manufacturer, and are shown inTable C. The average (Avg) from three separate measurements of thekinetic constants and binding constants of the parental Apu2.16 Fab, andfrom single measurements for the 24 affinity matured Fabs are shown.Neither the parental Apu2.16 Fab, nor any of the 24 affinity maturedFabs demonstrated detectable binding to K48-linked diubiquitin.

TABLE C Binding Constants for Affinity Matured Anti-K63-linkedPolyubiquitin Fabs Binding to K63-linked Diubiquitin as Measured by SPRclone kon (1/Ms) koff (1/s) Kd (nM) Apu2.16 Avg 4.9 × 10⁵ 5.4 × 10⁻² 110Standard 0.49 × 10⁵   2.0 × 10⁻² 30 deviation for Apu2.16 measurementsApu3.A8 1.7 × 10⁶ 1.4 × 10⁻² 8.7 Apu3.A9 2.3 × 10⁶ 9.9 × 10⁻² 44Apu3.A12 9.9 × 10⁹ 6.2 × 10¹   6.2 Apu3.B3 4.8 × 10⁶ 2.9 × 10⁻² 6.1Apu3.B5 1.0 × 10⁶ 4.6 × 10⁻² 46 Apu3.C1 4.3 × 10⁶ 7.8 × 10⁻² 18 Apu3.C44.7 × 10⁸ 3.4 × 10¹   73 Apu3.C5 1.0 × 10⁶ 2.9 × 10⁻² 28 Apu3.D3 7.9 ×10⁵ 6.1 × 10⁻² 78 Apu3.D7 8.1 × 10⁵ 3.7 × 10⁻² 46 Apu3.D8 3.9 × 10⁸ 4.4× 10⁰   11 Apu3.E1 1.1 × 10⁶ 2.1 × 10⁻² 18 Apu3.E2 1.0 × 10⁶ 3.0 × 10⁻²29 Apu3.E4 7.2 × 10⁵ 8.2 × 10⁻² 110 Apu3.E5 1.0 × 10⁶ 2.6 × 10⁻² 25Apu3.F1 8.3 × 10⁵ 2.3 × 10⁻² 28 Apu3.G1 6.5 × 10⁵ 5.4 × 10⁻² 84 Apu3.G26.1 × 10⁵ 5.4 × 10⁻² 90 Apu3.G3 1.0 × 10⁶ 4.1 × 10⁻² 39 Apu3.G4 7.3 ×10⁵ 2.4 × 10⁻² 33 Apu3.G5 1.1 × 10⁶ 3.1 × 10⁻² 29 Apu3.H5 1.0 × 10⁶ 2.9× 10⁻² 28 Apu3.H6 3.6 × 10⁹ 7.8 × 10¹   22 Apu3.H7 2.0 × 10⁴ 9.2 × 10⁻⁴45

(E) Western Blots of Affinity Matured Fabs

The binding constants obtained from the SPR analysis in Example 1(D)indicated that three clones (Apu3.A8, Apu3.A12, and Apu3.B3) displayedsingle digit nanomolar binding. These three Fabs along with the parentalApu2.16 Fab were tested for detection of K63-linked diubiquitin andK48-linked diubiquitin in a western blot. Six concentrations ofK63-linked diubiquitin (31-1000 ng) and three concentrations ofK48-linked diubiquitin (250-1000 ng) were run on a 4-12% NuPAGE gel(Invitrogen), transferred to a polyvinylidene fluoride (PVDF) membrane,and blotted with the parental clone and the three affinity-matured Fabs.The Fabs contained a carboxy-terminal 6×-His tag and were thus detectedwith an anti-pentaHis-HRP conjugated secondary antibody (Qiagen)followed by chemiluminescence development. All four Fabs specificallydetected K63-linked diubiquitin (see FIG. 3). No binding to K48-linkeddiubiquitin was observed for any of the four Fabs tested.

(F) Conversion to IgG

The parental Fab Apu2.16 and the three affinity-matured Fabs Apu3.A8,Apu3.A 12, Apu3.B3 were expressed in HEK293 cells as human IgGs.Expression constructs were generated by cloning the Fab variable domainsinto pRK mammalian expression constructs encoding the heavy and lightchains of human IgG (Gorman et al., DNA Prot. Eng. Tech. 2: 3-10(1990)). IgGs were purified by affinity chromatography on proteinA-sepharose columns by standard methodologies.

Example 2 Detection of Endogenously Ubiquitinated Proteins

The activity of the affinity-matured anti-K63-linked polyubiquitin IgGsdescribed in Example 1(F) was assessed. For western blot analyses,polyubiquitin or polyubiquitinated proteins were run on polyacrylamidegels and the contents of the gels were transferred to nitrocelluloseblots following standard procedures in the art. The resultingnitrocellulose blots were blocked for approximately an hour in 10 mMTris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-(TBST) containing 5% non-fatmilk powder. The primary anti-K63-linked polyubiquitin antibody (eitherthe parental Apu2.16 antibody in IgG format, or Apu3.A8, Apu3.A 12, orApu3.B3 in IgG format) was added to a final concentration of 5 μg/mL fora minimum of 1 hour at room temperature. Overnight incubations wereperformed at 4° C. The blots were washed three times in TBST, 10 minutesper wash. Bound anti-K63-linked polyubiquitin antibodies were detectedwith peroxidase-conjugated anti-human IgG (ICN Cappel) diluted 1:10,000in TBST containing 5% non-fat milk powder. After one hour at roomtemperature, the blots were washed 3-6 times in TBST, incubated inSupersignal (Pierce) according to the manufacturer's instructions, andexposed to film. For the western blots of endogenously ubiquitinatedproteins, 293T cells were transfected with or without a vectorexpressing 3×HA-tagged TRAF6 using Lipofectamine 2000 (Invitrogen).Cells were harvested two days post-transfection after culture in 25 μMMG-132 (Calbiochem) for the final hour. The cells were washed inphosphate-buffered saline and then protein extracts were prepared inice-cold lysis buffer (20 mM Tris-Cl pH 7.5, 135 mM NaCl, 1.5 mM MgCl₂,1 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 2mMN-ethyl-maleimide) supplemented with a complete protease inhibitorcocktail (Roche).

As shown in FIGS. 4 and 5, each of the three tested affinity-maturedanti-K63-linked IgGs worked better in western blots than the parentalApu2.16 IgG. FIG. 4 depicts the results of a western blot againstimmobilized purified K48-linked or K63-linked polyubiquitin containingtwo to seven ubiquitin subunits. The parental IgG Apu2.16 did not detectK63-linked or K48-linked polyubiquitin of any number of ubiquitinsubunits at any concentration tested. Affinity matured IgGs Apu3.A8,Apu3.A12, or Apu3.B3 detected K63-linked polyubiquitin containing 2 to 6subunits at each concentration tested, and of seven subunits at all butthe lowest concentration tested. No nonspecific binding to K48-linkedpolyubiquitin was observed with any of the three affinity-matured IgGs.To ascertain whether the affinity-matured antibodies were able to detectendogenously ubiquitinated proteins, 293T cells were transfected withTRAF6 and treated with MG-132, which has been found to result inK63-linked ubiquitination of TRAF6 in vivo (Wertz et al., Nature (2004)430: 694-699). Similar to the western blot with immobilized purifiedpolyubiquitin in FIG. 4, the parental Apu2.16 IgG was not able to detectK63-linked polyubiquitinated TRAF6 or any other K63-linkedpolyubiquitinated proteins in a western blot assay (FIG. 5). However,each of antibodies Apu3.A8, Apu3.A12, and Apu3.B3 specifically detectedK63-linked polyubiquitinated TRAF6 and other K63-linkedpolyubiquitinated proteins in the same western blot assay (FIG. 5).

The preceding experiments confirmed that the affinity-matured antibodieswere capable of detecting immobilized polyubiquitinated proteins.Further experiments were performed to ascertain whether these antibodieswere able to immunoprecipitate polyubiquitinated proteins. Forimmunoprecipitation assays, the previously described lysis buffer alsocontained 6 M urea and the cells were lysed at room temperature for 15minutes. Insoluble material was then removed by centrifugation and thesoluble lysate diluted in regular lysis buffer to lower the ureaconcentration to 0.29M. Lysates were precleared with protein A-sepharose(GE) for 1 hour at 4° C. and then incubated with 5 μg of the antibodyindicated for 1 hour at 4° C. Antibody complexes were captured withprotein A-sepharose at 4° C. for 1 hour, washed extensively in lysisbuffer, and eluted by boiling in Novex Tris-glycine SDS-sample buffer(Invitrogen) supplemented with 2.5% 2-mercaptoethanol.

When TRAF6-containing lysates were denatured in the presence of 1% SDSto dissociate it from other ubiquitinated proteins, andimmunoprecipitations were performed following dilution of the SDS to aslow as 0.05%, no TRAF6 was captured by the K63-specific antibodies (datanot shown). Immunoprecipitation of TRAF6 was successful, however, whenTRAF6-expressing cells were lysed in the presence of an alternatedenaturant: 6M urea. As shown in FIG. 6A, the three affinity-maturedantibodies immunoprecipitated K63-linked polyubiquitinated Traf6 betterthan the parental antibody Apu2.16, showing that the affinity-maturedantibodies were improved in binding to endogenously K63-ubiquitinatedproteins in solution as compared to the parental anti-K63-polybuiqutinantibody. In further experiments, treatment of the immunoprecipitatedK63-linked polyubiquitinated material with the promiscuousdeubiquitinating enzyme Usp2 for increasing periods of time collapsedthe smear of higher molecular weight bands observed in FIG. 6A and inthe second lane of FIG. 6B, confirming that the immunoprecipitated TRAF6was ubiquitinated (FIG. 6B, lanes 3-5). Non-specific proteolyticactivity potentially associated with Usp2 was unlikely to have beenresponsible for the observed disappearance of the slower migratingspecies because inhibition of Usp2 deubiquitinating activity using thecysteine protease inhibitor N-ethylmaleimide (NEM) prevented collapse ofthe smear (FIG. 6B, lane 6). A portion of the TRAF6 immunoprecipitatedby the K63-specific antibody Apu3.A8 appeared, by its migration, to beunmodified TRAF6, likely attributable to over-expression of TRAF6inducing the formation of self-oligomers that trap unmodified TRAF6.

These results were confirmed by mass spectrometry experiments. Whenpolyubiquitin chains are digested with trypsin, unique peptidescorresponding to each of the 7 different types of lysine linkage may beobserved. This follows the general consensus of a GlyGly motif on amiscleaved lysine. Each of the seven possible polyubiquitin chainlinkages yields a unique peptide with a unique mass (when measured at 10ppm mass accuracy) and also a unique fragmentation pattern uponcollision-induced dissociation. Therefore using a high resolution massspectrometer, along with targeted analysis against the specificsignature peptides of interest one can monitor and quantitate the levelsof abundance of either the K48 or K63 polyubiquitin chain linkage.Briefly, immunoprecipitation reactions from BJAB cells were performed asdescribed above using an anti-K48-linked antibody, a mixture of thethree affinity-matured anti-K63-linked polyubiquitin IgGs Apu3.A8,Apu3.A12, and Apu3.B3, or a control antibody (Herceptin®). Proteins wereextracted from the beads chemically using acetonitrile:water+0.1% TFA,reduced, alkylated, and digested with trypsin. A hybrid LTQ-Orbitrapmass spectrometer coupled to a nanoACQUITY™ HPLC (Thermo-FisherScientific) was used at a flow rate of 1 μL/minute with a one hourgradient (solvent A: water+0.1% formic acid; solvent B:acetonitrile+0.1% formic acid). The instrument was calibrated and tunedusing tryptically digested synthetic tetraubiquitin (Boston Biochem) tovalidate optimum charge states for targeted analysis studies. Thedetermined m/z for the K48-linked polyubiquitin were 487.6, 730.89, and1460.78, and the determined m/z for the K63-linked polyubiquitin were561.80, 748.73, and 1122.6. After tryptic digestion, reverse phaseseparation and targeted tandem mass spectrometry the ion chromatogramsfor the K48 and K63 polyubiquitin peptides were extracted and the peakarea was calculated to infer concentration.

The results of the immunoprecipitation analyses are set forth in FIGS.7A and 7B. As shown in FIG. 7A, the mixture of the threeaffinity-matured anti-K63-linked polyubiquitin antibodies specificallyimmunoprecipitated K63-linked polyubiquitinated proteins. The results inFIG. 7B show that the anti-K63-linked polyubiquitin antibodies are notcross-reactive for K48-linked polyubiquitin peptides (whereas forcomparison the K48-linked polyubiquitin antibody shows substantialenrichment for the K48-linked polyubiquitin peptides).

In further mass spectrometry experiments, polyubiquitinated proteinswere immunoprecipitated from human BJAB cells as described above, andthen resolved by SDS-PAGE using standard methods. The immunoprecipitatedgel-resolved proteins were subjected to in-gel trypsin digestion andthen analyzed using a variation of the ubiquitin-AQUA method(Kirkpatrick et al., Nat. Cell Biol. 8: 700-710 (2006)). Briefly, thetrypsin digests were supplemented with isotope-labeled internal standardpeptides representing each polyubiquitin chain linkage and unbranchedubiquitin, and then the standards plus their corresponding nativeanalytes were detected in high-resolution precursor ion scans usingnarrow range-extracted ion chromatograms (Crosas et al., Cell 127:1401-1413 (2006)). The abundance of each polyubiquitin chain linkage andthe total amount of ubiquitin in the BJAB immunoprecipitates wasquantified by comparing the signal from each digested peptide relativeto its corresponding internal standard. Specifically, BJAB cells werecultured in spinner flasks in the high-glucose version of Dulbecco'smodified Eagle's medium supplemented with 100 μM L-asparagine, 50 μM2-mercaptoethanol, and 10% fetal calf serum. Cells (>10⁹) were washed inPBS and lysed for 30 minutes at room temperature in lysis buffer (20 mMTris-HCl pH 7.5, 135 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100,10% glycerol) containing 6 M urea, 2 mM N-ethylmaleimide (NEM) and acomplete protease inhibitor cocktail (Roche). Insoluble material wasremoved by centrifugation and then the lysate was diluted to 4 M ureawith lysis buffer supplemented with protease inhibitors, 2 mM NEM and 1mM DTT. After preclearing with Protein A-sepharose (GE Healthcare) forone hour, the lysate was divided into three samples that received 30 μgof either Apu3.A8 anti-K63 (in IgG format), Apu2.07 anti-K48 (in IgGformat), or isotype control antibody (anti-Her2). Samples were incubatedat room temperature for two hours and subsequently centrifuged to removeprecipitated material. Further antibody (10 μg) was added to the solublelysate as well as Protein A-sepharose and samples were incubated at roomtemperature overnight. The sepharose beads were washed extensively inlysis buffer, then in PBS. The beads were eluted in reducing SDS samplebuffer, alkylated with iodoacetamide, and then resolved by SDS-PAGE in4-20% Tris glycine gels (Invitrogen). All areas of the gel (except forthe 25 and 55 kDa regions representing the heavy and light chains fromthe precipitating antibodies) were excised, crushed, washed in 25 mMNH₄HCO₃ in 50:50 acetonitrile:water and dehydrated. The gel was thenrehydrated in 25 mM NH₄HCO₃ containing trypsin (Promega) and incubatedovernight at 37° C. The reaction was quenched by addition of 0.008% TFA.Peptides were extracted in 5% acetic acid and then twice in 100%acetonitrile. These samples were spiked with AQUA peptide standards (1pmol) and injected via an auto-sampler for separation by reverse phasechromatography on a NanoAcquity HPLC system (Waters). Peptides loadedonto a pre-column (5 μm Symmetry® C18, 180×20 mm) were separated usingan analytical column (1.7 μm BEH-130 C18 column 100×100 mm (Waters))with a flow rate of 1 μL per minute and a gradient of 2% solvent B to90% Solvent V (where Solvent A is water+0.1% formic acid and solvent Bis 100% acetonitrile+0.1% formic acid) applied over 70 minutes with atotal analysis time of 90 minutes. Peptides were eluted directly into ananospray ionization source with a spray voltage of 2 kV and wereanalyzed using an LTQ XL-Orbitrap mass spectrometer (ThermoFisher).Precursor ions were analyzed in the FTMS at 60,000 resolution.Quantitation was performed by comparing peak areas for the heavy andlight version of each peptide, extracting the ion chromatograms at 10ppm mass accuracy to 4 decimal places.

MuRF1 autoubiquitination reactions were performed according to themanufacturer's instructions (Boston Biochem). For immunoprecipitations,samples were diluted 100-fold in lysis buffer containing 4 M urea,precleared with Protein A-sepharose, and then immunoprecipitatedovernight with antibody (20 μg) and Protein A-sepharose. Total ubiquitinwas blotted with P4D1 antibody (Santa Cruz Biotechnology). MuRF1 samplesanalyzed by mass spectrometry were resolved on an Agilent 1100 LC modulewith a 15 minute gradient from 5% solvent B to 30% Solvent B and a totalanalysis time of 30 minutes. Ion chromatograms were created byextracting a window ±15 ppm from the expected m/z.

Anti-K48-linked polyubiquitin antibody Apu2.07, the anti-K63-linkedpolyubiquitin antibody Apu3.A8 and an isotype control antibodyrecognizing Her2 immunoprecipitated 49.1 pmol, 2.2 pmol and 0.2 μmol oftotal ubiquitin, respectively (FIG. 7C). This result was in keeping withthe observation that K48-linked polyubiquitin chains were more abundantthan K63-linked polyubiquitin chains in the input lysate used for theimmunoprecipitations (FIG. 7D). Direct examination of the polyubiquitinlinkages immunoprecipitated by the anti-K48 antibody revealed 12.7 pmolof K48-GG signature peptide and residual amounts of the signaturepeptides for certain other linkages (0.1 pmol K63, 1.1 pmol K11, and 2.0pmol K6) (FIG. 7E). Similarly, polyubiquitin chains immunoprecipitatedby the anti-K63 linked polyubiquitin antibody Apu3.A8 mainly producedthe K63-GG signature peptide (0.3 pmol K63), and lesser amounts of otherlinkage peptides (0.18 pmol K48, 0.08 pmol K11, and 0.06 pmol K6) (FIG.7F). Given the strong binding preferences seen for theanti-polyubiquitin antibodies by surface plasmon resonance, these massspectrometry results suggest that a significant fraction of cellularsubstrates modified by ubiquitin exhibit heterogeneous polyubiquitinchain linkages.

To confirm that these “off-target” linkages immunoprecipitated from BJABcell lysates were derived from substrates bearing heterogeneouspolyubiquitin chains, as opposed to non-specific binding, the necessityof the target linkage for immunoprecipitation of the “off target”linkages was investigated. Mixed linkage polyubiquitin chains lackingeither the K48 or the K63 linkage were generated in vitro using E3 MuRFIin combination with the promiscuous E2 enzyme UbCH5c (Kim et al. J.Biol. Chem. 282: 17375-17386 (2007)). The K48 or K63 linkage wasexcluded from the MuRFI autoubiquitination reactions using mutantubiquitin that had either K48 or K63 mutated to arginine (FIG. 7G). Bywestern blotting with a pan-ubiquitin antibody, MuRF-1autoubiquitination reactions produced high molecular weight smearsirrespective of whether wild-type, K48R, or K63R ubiquitin was used(FIG. 7H, lanes 1, 5, and 9). Ubiquitin-AQUA analysis of the reactionwith wild-type ubiquitin revealed three main linkages: K48, K63, and K11(FIG. 7I, lane 1). As expected, reactions performed with K48R ubiquitinlacked K48-linked polyubiquitin chains (FIG. 7I, lane 9). Both theanti-K48-linked and anti-K63-linked polyubiquitin antibodiesimmunoprecipitated polyubiquitinated species when MuRF1 was modifiedwith wild-type ubiquitin (FIG. 7H, lanes 3-4), but each antibody failedto do so if the target linkage for which it was specific was absent(FIG. 7H, lanes 7 and 12). An isotype-matched control antibody(anti-HER2) did not immunoprecipitate ubiquitinated MuRFI from any ofthe reactions (FIG. 7H, lanes 2, 6, and 10). Ubiquitin-AQUA analysis ofthe immunoprecipitated species form the wild-type Ub reactiondemonstrated the presence of K48-, K63-, and K11-linked chainsregardless of the antibody used for enrichment (FIG. 7J, lane 3 and FIG.7K, lane 4). As predicted by Western blotting (FIG. 7H, lanes 7 and 12),K11-linked chains were immunoprecipitated by both the anti-K48-linkedand the anti-K63-linked polyubiquitin antibodies only if their targetlinkage was also present (FIGS. 7J and 7K). These results demonstratedthat the anti-K48-linked and anti-K63-linked polyubiquitin antibodiesretain their fidelity in the context of immunoprecipitation. Further,they indicate that alternative linkages immunoprecipitated from cells bythe anti-K48-linked and anti-K63-linked polyubiquitin antibodies muststem from individual substrates bearing both target and off-targetlinkages.

Example 3 Ubiquitination Pathway Detection Using Affinity-MaturedAntibodies

A number of cellular pathways have been identified that are regulated bypolyubiquitination of key proteins. The affinity-matured anti-K63-linkedpolyubiquitin antibodies of the invention were used to determine theextent of K63-linked polyubiquitination of particular proteins in thecell, helping to elucidate cellular signaling pathways whereubiquitination with K63-linked polyubiquitin plays a role.

(A) TNFα Activation of TNFR1

TNFα activation of tumor necrosis factor receptor 1 (TNFR1) leads to RIPand TRAF2 association (see FIG. 8A). TRAF2 adds K63-linked polyubiquitinchains to RIP which allows recruitment of TAK1/TAB2/TAB3 and subsequentactivation of the NFκB signaling pathway (see Wertz et al., Nature(2004) 430: 694-699). Downregulation of signaling through this pathwayoccurs by the deubiquitinase A20 removing the K63-linked polyubiquitinchains on RIP and replacing them with K48-linked polyubiquitin chains,targeting RIP to the proteasome for degradation. The affinity maturedanti-K63-linked polyubiquitin antibodies of the invention were used toassess the degree and type of polyubiquitination of RIP during variousperturbations of the pathway.

Briefly, HeLa S3 cells were treated with 21 μM MG-132 for ten minutes.The cells were subsequently treated with 100 ng/mL TNF from 0 to 25minutes. At each timepoint, the cells were pelleted and washed once withPBS. The washed pellet was lysed in 20 mL TNFR1 immunoprecipitationbuffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA)including protease inhibitors, 25 μM MG-132, 10 mM N-ethyl maleimide(NEM), and 50 mM NaF for 10 minutes at 4° C. with rotation. Lysates werecentrifuged at 10,000×g for 5 minutes. The cleared lysate was incubatedwith 200 μL Protein A beads for one hour at 4° C. with rotation. Beadsand debris were pelleted by centrifugation at 2,000 rpm for 5 minutes.Samples were taken from each timepoint lysate for western blot analysisas described above. For immunoprecipitation samples, 20 μL anti-TNFR1antibody was added to each sample, and the samples were rotated at 4° C.for 2.5 hours. 200 μL unblocked protein A beads were added to eachsample, and the samples were again rotated at 4° C. for 2.5 hours. Thebeads were washed twice with immunoprecipitation buffer, twice inimmunoprecipitation buffer including 1 M NaCl, and twice inimmunoprecipitation buffer. Proteins specifically bound to the beadswere recovered by treatment with ubiquitin chain lysis buffer (20 mMTris-Cl pH 7.5, 135 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100and 10% glycerol) containing 6 M urea and gentle agitation for 15minutes at room temperature. The beads were pelleted by centrifugation.Supernatants were diluted 25-fold with ubiquitin chain lysis bufferincluding protease inhibitors, 10 mM NEM and 0.5 mM dithiothreitol,followed by preclearing with 50 μL Protein A beads+10 μg Herceptin for 2hours. Anti-K48-linked polyubiquitin antibodies and anti-K63-linkedpolyubiquitin antibodies (1:1 mixture of Apu3.A8 and Apu3.B3) werepre-coupled to protein A beads for 3 hours at 4° C. Immunoprecipitationreaction samples were combined with one of the precoupled antibodies androtated for 2 hours at 4° C., followed by washing in lysis buffer andthe addition of sample buffer. Samples were reduced and alkylated andrun on 4-12% Tris/Gly 1.5 mm 15-well Novex gels, followed by transfer,immunoblotting, and western blot analysis according to the proceduresdescribed above in Example 2.

A simplified form of the known RIP modification pathway is shownschematically in FIG. 9B. Treatment of HeLa S3 cells with TNFαstimulates K63-linked polyubiquitination of RIP, which is in a complexwith TNFR1. A two-stage immunoprecipitation (first immunoprecipitatingthe TNFR1 complex, then immunoprecipitating K48-linked polyubiquitinatedproteins, or K63-linked polyubiquitinated proteins) permitted ananalysis of the ubiquitination state of RIP upon TNFα treatment. Asdepicted in FIG. 9A, RIP protein quickly associates with TNFR1 withinthe first five minutes of TNFα treatment, and the amount of associatedRIP remains constant regardless of the time of TNFα treatment thereafter(blot 4). The RIP that is initially associated with TNFR1 is K63-linkedpolyubiquitinated, but longer treatment with TNFα leads to chainediting, resulting in K48-linked polyubiquitinated RIP (compare blots 6and 7). Thus the ability to discriminate between RIP bearing aK63-linked polyubiquitin label versus RIP bearing a K48-linkedpolyubiquitin label provides important information about a cellularpathway triggered by TNFα, and the affinity-matured antibodies of theinvention provide a convenient and useful tool for examining thisRIP-mediated pathway without performing mass spectrometry or otherbiophysical analyses.

(B) Polyubiquitin Chain Editing of IRAK1

Given that the kinase RIP1 is modified sequentially by K63- and thenK48-linked polyubiquitin chains, the latter being conferred by the E3deubiquitinase A20, and that A20 is also a known negative regulator ofsignaling by certain TLRs (Boone et al. Nat. Immunol. 5: 1052-1060(2004)), other kinase adaptors recruited to TLRs and the interleukin-1receptor (IL-1R) were investigated for regulation in a similar fashion.IRAK1 was a potential candidate for undergoing polyubiquitin chainediting because post-translationally modified IRAK is recruited rapidlyto activated receptor complexes and subsequently is degraded (Yamin andMiller J. Biol. Chem. 272: 21540-21547 (1997)). 293 cells stablyexpressing IL-1R and IL1R-AcP were treated with 25 μM MG-132 just priorto the addition of 10 ng/mL IL-1 (eBIoscience). Cells were washed withPBS and lysed in IRAK IP buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 1.5 mMMgCl₂, 2 mM EGTA, 10 mM NaF, 2 mM DTT, and 0.5% Triton X-100) containing6 M urea. Soluble lysates were diluted approximately 20-fold in lysisbuffer and immunoprecipitations with either anti-K48-linkedpolyubiquitin antibody Apu2.07 or a 1: mix of anti-K63-linkedpolyubiquitin antibody Apu3.A8 and Apu3.B3 were performed as describedabove in Example 3(A) for RIP1. The IRAK1 antibody was obtained fromSanta Cruz Biotechnology.

Upon treatment with IL-1β, cells stably expressing the IL-1R revealed asmear of modified IRAK1 within two minutes (FIG. 10A). Theslower-migrating forms of IRAK1 were more abundant at 10 minutes andcould be converted to the unmodified form of IRAK1 by Usp2 in anNEM-sensitive manner. These results suggested that the higher molecularweight forms of IRAK1 are due to ubiquitination. The appearance ofubiquitinated IRAK1 coincided with activation of downstream NF-κBsignaling as evidenced by IκBA degradation (FIG. 10B). At differenttimes after treatment with IL-11, cell lysates were prepared in 6 M ureaand immunoprecipitated with linkage-specific polyubiquitin antibodiesprior to Western blotting for IRAK1 (FIG. 10C). IRAK1 modified withK63-linked polyubiquitin chains was detected at 5 minutes after IL-1βtreatment and was maximal by 30-60 minutes post treatment, mirroring thetiming of appearance of the high molecular weight IRAK1 smear (comparethe upper panel of FIG. 10B with the lower panel of FIG. 10C). Theamount of K48-linked polyubiquitin evident on IRAK1 peaked at 90-150minutes, after which time the intensity of the total IRAK1 signal beganto subside. When cells were treated with IL-1β in the presence of theproteasome inhibitor MG-132, this degradation of ubiquitinated IRAK1 wasinhibited (FIG. 10D), consistent with the K48-linked polyubiquitinchains targeting IRAK1 for proteasomal degradation. Thus polyubiquitinchain editing of IRAK1 in cells treated with IL-1β resembles that ofRIP1 in cells treated with TNF and this process of polyubiquitin chainediting may represent a general mechanism of terminating downstreamsignaling events. As described above for RIP, the ability todiscriminate between IRAK1 bearing a K63-linked polyubiquitin labelversus IRAK1 bearing a K48-linked polyubiquitin label provides importantinformation about a cellular pathway triggered by IL-1β (see FIG. 8B),and the affinity-matured antibodies of the invention provide aconvenient and useful tool for examining this IRAK1-mediated pathwaywithout performing mass spectrometry or other biophysical analyses.

Example 4 Crystal Structure of Apu3.A8 in Complex with K63dUB

To understand the structural consequences of the observed affinity andspecificity improvements in Apu3.A8, the Fab fragment was crystallizedin complex with K63-linked diubiquitin and the structure was solved to2.6 Å resolution (FIG. 2C). Briefly, the Fab fragment of Apu3.A8 wasexpressed in E. coli, purified using Protein G-sepharose, and elutedwith 0.58% acetic acid. Fab-containing fractions were purified using anSP HiTrap column (GE Healthcare) equilibrated in 20 mM MES pH 5.5 andeluted with an NaCl gradient. The protein was further purified bypassing it over an S-200 column (GE Healthcare) in 20 mM Tris-HCl pH7.3, 150 mM NaCl. K63-linked diubiquitin was produced by incubating 4.1mM ubiquitin (K63R and D77), 0.1 μM E1 (Boston Biochem), and 20 μM E2(UbcH13/Uev1a for K63) (Boston Biochem) in 50 mM Tris-HCl, 5 mM MgCl₂,0.5 mM DTT, 2.5 mM ATP overnight at 37° C. (see, e.g., Pickart andRaasi, Meth. Enzymol. 399: 21-36 (2005)). Diubiquitin was purified usinga MonoS cation exchange column in a NaCl gradient and 20 mM MES pH 5.5,and then concentrated to 0.5 mg/mL. Diubiquitin-Fab complexes wereprepared from 3-fold molar excess of Fab with diubiquitin. The proteincomplex was purified over a Superdex 75 column in 20 mM Tris-HCl pH 7.3,150 mM NaCl. Complex fractions were pooled and concentrated to 17 mg/mLfor crystallization screening trials. Crystals of K63Rdiubiquitin-Apu3.A8 (90 μm×90 μm×150 μm) grew after two weeks at 18° C.in sitting drops from 1:1 mixtures of protein (17 mg/mL in 20 mMTris-HCl pH 7.3, 150 mM NaCl) and well solution (0.1 M Tris-HCl pH 8.0,1.6 M LiSO₄). Crystals were cryoprotected using well solution with theaddition of 30% glycerol. Crystallographic data was collected at SSRLbeamline 7-1 (Table D) and was processed with HKL (HKL). The structurewas solved by molecular replacement using the program PHASER (CCP4)followed by refinement with REFMAC5 (CCP4). The Apu3.A8 complex wassolved using the refined Apu2.16 complex as a search model (see FIG. 2Aand US Patent Publication No. US2007-0218069) (FIGS. 2C and 2D). Allfigures were made with PyMol (www.pymol.org).

TABLE D X-Ray Data Collection and Refinement Statistics Ubq-Apu2.16Ubq-Apu3A8 Data collection Space group C2 C2 Cell dimensions a, b, c (Å)177, 95, 98 107, 88, 90 β (°) 107 108 Resolution (Å) 50-2.7 (2.80-2.70)50-2.6 (2.69-2.60) R_(sym) 5.0 (52.6) 5.1 (58.0) <I/σI> 15.9 (1.9) 11.6(1.9) Completeness (%) 99.2 (100) 98.1 (97.9) Redundancy 3.8 (3.9) 3.8(3.9) Refinement Resolution (Å) 30-2.7 50-2.6 Complex in asu 2 1 No.reflections 40,137 21,569 R_(work)/R_(free) (%) 22.1, 27.3 24.0, 30.1No. atoms Protein 8914 4469 Solvent 0 25 R.m.s. deviations Bond lengths(Å) 0.009 0.009 Bond angles (°) 1.2 1.1 *Values in parentheses are forhighest-resolution shell.

The resulting structure showed that the changes in L2 and H2, which hadgreatly improved the specificity, had relatively little impact on thestructure of the Fab (FIG. 2C). Excluding differences in theconformation of CDR L3 and the N-terminal strand of the light chain,both of which are attributable to differences in crystal packing, thestructure of the A8 Fab is virtually identical to the structure of theparental A16 Fab (rmsd 0.08 Å on 412 C-α atoms). Both structures werecompared to the Fv region of the humanized anti-HER2 antibody 4D5 (FIG.2D). The conformation of L1 and the N-termini of the three Fabs differconsiderably. The C-alpha atom of T5 is displaced by 14 Angstromsbetween the structures of either Apu2.16 or Apu3.A8 and humanized 4D5,despite identical sequences among the three structures in these regions.The conformation and sequence of L3 differs considerably as well. Thesedifferences combined to affect the position of Q90 in Apu3.A8 such thatit occupies the space normally occupied by the N-terminal strand,resulting in displacement of this strand with concomitant rearrangementof L1. Comparison of the structure of K63 diubiquitin in the aboveco-crystal structure that of a crystal structure of K63-diubiquitinalone (pdb code 2JF5) or to either the crystal or solution structures ofK48 diubiquitin (Cook et al., J. Biol. Chem. 267: 16467-16471 (1992);Varadan et al., J. Mol. Biol. 324: 637-647 (2002)) demonstrates that theFabs recognize a specific conformation of diubiquitin that is unique tothe K63 linkage.

Surprisingly, neither R52 in L2 nor T52 in H2 is in intimate contactwith the diubiquitin, and neither residue contributes much to theubiquitin-binding surface on Apu3.A8. Instead, the effect of these twomutations appears to be driven largely by improved electrostaticcompatibility between the surface of the K63-acceptor ubiquitin and thelight chain (FIG. 2E). In the Apu3.A8 light chain, R52 (which wasintroduced in Apu3.A8) and R66 contribute to a positive region that isin close proximity to a negatively charged region on the ubiquitinsurface, created in part by residues D21, D58, and E18 from theK63-acceptor ubiquitin.

Example 5 Localization of Polyubiquitin Chains within Cells

The preceding experiments demonstrate that the affinity-maturedantibodies of the invention are capable of sensitive and specificdetection of K63-linked polyubiquitinated proteins in the context of awestern blot, are able to specifically immunoprecipitate K63-linkedpolyubiquitinated proteins from cellular lysates, and can be used toassist in the elucidation of cellular pathways involving K63-linkedpolyubiquitination of one or more proteins. Further experiments wereperformed to ascertain the ability of the antibodies be used in indirectimmunofluorescence microscopy for visualization of polyubiquitinatedspecies within cells, and where any such detected proteins localize.HeLa cells grown in 2-well chamber slides were fixed with 4%paraformaldehyde in PBS at room temperature for 20 minutes, rinsed twicewith PBS, and permeabilized with PBS containing 0.1% Triton X-100 for 5minutes. After blocking in Earle's balanced salt solution supplementedwith 10% goat serum, 0.1% Triton X-100 and 0.1% saponin for one hour,cells were labeled overnight at 4° C. with 1 μg/mL Apu2.07 anti-K48, 1μg/mL Apu3.A8 anti-K63, or 5 μg/mL PW8155 anti-proteasome antibodies(Biomol International) in blocking solution. Cells were washed threetimes with PBS containing 0.1% Triton X-100 and then incubated with 4drops of Image-IT Fx signal enhancer (Invitrogen) for 15 minutes. Cellswere washed three times with PBS containing 0.1% Triton X-100, thenstained for 1 hour with Cy2-conjugated anti-human and TexasRed-conjugated anti-rabbit antibodies (Jackson ImmunoResearch) dilutedin blocking solution. Cells were washed three times with PBS containing0.1% Triton X-100, rinsed twice with water, and then mounted withProLong GoId™ containing DAPI (Invitrogen) and a 1.5 mm coverslip. Cellswere examined under an Axioplan 2 light microscope (Zeiss) and imageswere recorded with a CooISNAP_(HQ) CCD camera (Photometrics) controlledby SlideBook™ software (Intelligent Imaging Innovations). In addition,Z-series images were acquired and analyzed by deconvolution microscopy,using SlideBook™ or AutoQuant™ (Media Cybernetics) software (data notshown). To confirm the localization of the signal, samples were alsoexamined with an LSM510 META laser-scanning confocal microscope (Zeiss).All of the images presented are single-capture wide-field fluorescentmicrographs.

The results are shown in FIG. 11. The anti-K48-linked polyubiquitinantibody Apu2.07 stained both the nucleus and cytoplasm of HeLa cells,but did not label nucleoli (FIG. 11A). This pattern of stainingcoincided almost completely with proteasomes labeled with a polyclonalantibody recognizing the core 20S subunits (FIGS. 11B and C), consistentwith the idea that K48-linked polyubiquitin chains generally targetproteins for proteasomal degradation. K48-linked polyubiquitin chains,but not proteasomes, were detected at the midbody during mitosis. Incontrast, HeLa cells stained with the anti-K63-linked polyubiquitinantibody Apu3.A8 labeled cytoplasmic speckles that varied both in numberand size between individual cells (FIG. 1 ID). No co-localization ofK63-linked polyubiquitin chains with proteasomes was detected in thosespeckles (FIGS. 11E and F). In control experiments, staining by thelinkage-specific antibodies could be competed by synthesizedpolyubiquitin chains only if the target linkage was present (data notshown). These results indicate that K48- and K63-linked polyubiquitinchains can occur in distinct subcellular regions, and that antibodiesApu2.07 and Apu3.A8 are able to detect K48-linked or K63-linkedpolyubiquitin in indirect immunofluorescence assays.

1. (canceled)
 2. An isolated antibody or antigen-binding fragmentthereof that specifically binds to K63-linked polyubiquitin, comprisingat least one hypervariable (HVR) sequence selected from HVR-H2 andHVR-L2 of any of SEQ ID NOs: 59-110 and 112, and 7-58 and 111,respectively.
 3. An isolated antibody or antigen-binding fragmentthereof that specifically binds to K63-linked polyubiquitin, comprisingat least one HVR-H2 sequence, wherein HVR-H2 comprises the amino acidsequence a b c d e f g h i j k l m n o p (SEQ ID NO: 221), wherein aminoacid a is selected from the amino acids tyrosine, aspartic acid andtryptophan; amino acid b is isoleucine; amino acid c is selected fromthe amino acids serine, threonine, alanine, phenylalanine, tyrosine, andvaline; amino acid d is proline; amino acid e is tyrosine; amino acid fis selected from the amino acids tyrosine, phenylalanine, leucine, andhistidine; amino acid g is glycine; amino acid h is selected from theamino acids serine, glycine, alanine, phenylalanine, and tryptophan;amino acid I is threonine; amino acid j is serine; amino acid k istyrosine; amino acid l is alanine; amino acid m is aspartic acid; aminoacid n is serine; amino acid o is valine, and amino acid p is lysine. 4.The antibody or antigen-binding fragment of claim 2, comprising at leastone HVR-H2 sequence selected from the HVR-H2 sequences of SEQ ID NOs:59-110 and
 112. 5. The antibody or antigen-binding fragment of claim 2,comprising at least one HVR-H2 sequence selected from the HVR-H2sequences of SEQ ID NOs: 60, 63, and
 66. 6. An isolated antibody orantigen-binding fragment thereof that specifically binds to K63-linkedpolyubiquitin, comprising at least one HVR-L2 sequence, wherein HVR-L2comprises the amino acid sequence q r s t u v w x (SEQ ID NO: 222),wherein amino acid q is selected from the amino acids tyrosine andphenylalanine; amino acid r is selected from the amino acids alanine andserine; amino acid s is alanine; amino acid t is selected from the aminoacids serine, arginine, valine, threonine, alanine, asparagine, andleucine; amino acid u is serine; amino acid v is leucine; amino acid wis tyrosine, and amino acid x is serine.
 7. The antibody orantigen-binding fragment of claim 2, comprising at least one HVR-L2sequence selected from the HVR-L2 sequences of SEQ ID NOs: 7-58 and 111.8. The antibody or antigen-binding fragment of claim 2, comprising atleast one HVR-L2 sequence selected from the HVR-L2 sequences of SEQ IDNOs: 8, 11, and
 14. 9. (canceled)
 10. The antibody or antigen-bindingfragment of claim 3, further comprising at least one HVR-L2 sequence,wherein HVR-L2 comprises the amino acid sequence q r s t u v w x (SEQ IDNO: 222), wherein amino acid q is selected from the amino acids tyrosineand phenylalanine; amino acid r is selected from the amino acids alanineand serine; amino acid s is alanine; amino acid t is selected from theamino acids serine, arginine, valine, threonine, alanine, asparagine,and leucine; amino acid u is serine; amino acid v is leucine; amino acidw is tyrosine, and amino acid x is serine.
 11. The antibody orantigen-binding fragment of claim 2, comprising at least one HVR-H2sequence selected from the HVR-H2 sequences of SEQ ID NOs: 59-110 and112 and at least one HVR-L2 sequence selected from the HVR-L2 sequencesof SEQ ID NOs: 7-58 and
 111. 12. The antibody or antigen-bindingfragment of claim 2, comprising at least one HVR-H2 sequence selectedfrom the HVR-H2 sequences of SEQ ID NOs: 60, 63, and 66 and at least oneHVR-L2 sequence selected from the HVR-L2 sequences of SEQ ID NOs: 8, 11,and
 14. 13. The antibody or antigen-binding fragment of claim 2, whereinthe antibody or antigen-binding fragment comprises HVR amino acidsequences selected from the HVR-H2 and HVR-L2 amino acid sequences setforth for any one of Apu3.A8-Apu3.H10 in Table B.
 14. The antibody orantigen-binding fragment of claim 2, wherein the antibody orantigen-binding fragment comprises HVR amino acid sequences selectedfrom the HVR-H2 and HVR-L2 amino acid sequences set forth for any one ofApu3.A8, Apu3.A12, and Apu3.B3 in Table B.
 15. The antibody orantigen-binding fragment of claim 2, comprising at least one HVRsequence selected from the HVR-H1 sequence of SEQ ID NO: 5, the HVR-H3sequence of SEQ ID NO: 6, the HVR-L1 sequence of SEQ ID NO: 3 and theHVR-L3 sequence of SEQ ID NO:
 4. 16. (canceled)
 17. (canceled) 18.(canceled)
 19. The antibody or antigen-binding fragment of claim 2,wherein the affinity of the antibody or antigen-binding fragment forK63-linked polyubiquitin is improved relative to the affinity of theparental Fab Apu2.16.
 20. The antibody or antigen-binding fragment ofclaim 19, wherein the K_(d) of the antibody or antigen-binding fragmentfor K63-linked polyubiquitin is less than or equal to 10 nM.
 21. Anisolated antibody or antigen-binding fragment that binds to the sameantigenic determinant on polyubiquitin as the antibody orantigen-binding fragment of claim 2, wherein the antibody orantigen-binding fragment does not specifically bind to K48-linkedpolyubiquitin or monoubiquitin.
 22. An isolated antibody orantigen-binding fragment that competes with the antibody of claim 2 forbinding to polyubiquitin, wherein the antibody or antigen-bindingfragment does not specifically bind to monoubiquitin.
 23. The antibodyor antigen-binding fragment of claim 2, wherein the antibody orantigen-binding fragment specifically binds to a K63-linkedpolyubiquitinated protein.
 24. The antibody or antigen-binding fragmentof claim 23, wherein the antibody or antigen-binding fragment has atleast one property selected from inhibiting degradation of theK63-linked polyubiquitinated protein, modulating at least onepolyubiquitin-mediated signaling pathway, inhibiting at least onepolyubiquitin-mediated signaling pathway, and stimulating at least onepolyubiquitin-mediated signaling pathway.
 25. (canceled)
 26. (canceled)27. (canceled)
 28. A nucleic acid molecule encoding the antibody orantigen-binding fragment of claim
 2. 29. A vector that comprises thenucleic acid of claim
 28. 30. A host cell comprising the vector of claim29
 31. A cell line capable of producing the antibody or antigen-bindingfragment of claim
 2. 32. A method of producing the antibody orantigen-binding fragment of claim 2, comprising culturing a host cellcomprising a nucleic acid molecule encoding the antibody orantigen-binding fragment under conditions wherein the antibody orantigen-binding fragment is produced.
 33. A composition comprising aneffective amount of the antibody or antigen-binding fragment of claim 2and a pharmaceutically acceptable carrier.
 34. A method of identifyingthe presence of K63-linked polyubiquitin or a K63-linkedpolyubiquitinated protein in a sample, comprising contacting the samplewith at least one antibody or antigen-binding fragment of claim
 2. 35. Amethod for the treatment of a disease or condition associated withdysregulation of polyubiquitin in a patient, the method comprisingadministering to the patient an effective amount of at least oneantibody or antigen-binding fragment of claim
 2. 36. (canceled)
 37. Themethod of claim 35, wherein the patient is human.
 38. The method ofclaim 35, wherein the disease is selected from cancer, a musculardisorder, a ubiquitin-pathway-related genetic disorder, animmune/inflammatory disorder, and a neurological disorder.
 39. Themethod of claim 38, wherein the disease is selected from carcinoma,lymphoma, blastoma, sarcoma, leukemia, muscular dystrophy, multiplesclerosis, amyotrophic lateral sclerosis, cystic fibrosis, Angelman'ssyndrome, Liddle syndrome, Alzheimer's disease, Parkinson's disease,Pick's disease, and Paget's disease.
 40. A method of determining thepresence of a K63-linked polyubiquitin or a K63-linked polyubiquitinatedprotein in a sample suspected of containing a K63-linked polyubiquitinor K63-linked polyubiquitinated protein, comprising exposing the sampleto at least one antibody or antigen-binding fragment of claim 2 anddetermining the binding of the at least one antibody or antigen-bindingfragment to a K63-linked polyubiquitin or K63-linked polyubiquitinatedprotein in the sample.
 41. A method of separating K63-linkedpolyubiquitinated protein from non-K63-linked polyubiquitinated proteinin a sample, comprising contacting the sample with at least one antibodyor antigen-binding fragment of claim
 2. 42. A method of determining thefunction and/or activity of K63-linked polyubiquitin in a cellcomprising contacting the cell with at least one antibody orantigen-binding fragment of claim 2 and assessing the effect of saidcontacting step on the cell.
 43. A method of determining the functionand/or activity of K63-linked polyubiquitin in a sample comprisingcontacting the sample with at least one antibody or antigen-bindingfragment of claim 2 and assessing the effect of said contacting step onthe sample.